Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells.

Purpose: Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. This study delineates how osteosarcoma cells educate the lung microenvironment during metastatic progression. Experimental design: Using single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence. We evaluated the ability of nintedanib to impair metastatic colonization and prevent osteosarcoma-induced education of the lung microenvironment in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Results: Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially-differentiated epithelial intermediates. Inhibition of fibrotic pathways with nintedanib prevented metastatic progression in multiple murine and human xenograft models. Conclusions: Our work demonstrates that osteosarcoma cells co-opt fibrosis to promote metastatic outgrowth. When harmonized with data from adult epithelial cancers, our results support a generalized model wherein aberrant mesenchymal-epithelial interactions are critical for promoting lung metastasis. Adult epithelial carcinomas induce fibrotic pathways in normal lung fibroblasts, whereas osteosarcoma, a pediatric mesenchymal tumor, exhibits fibrotic reprogramming in response to the aberrant wound-healing behaviors of an otherwise normal lung epithelium, which are induced by tumor cell interactions. Statement of translational relevance: Therapies that block metastasis have the potential to save the majority of lives lost due to solid tumors. Disseminated tumor cells must educate the foreign, inhospitable microenvironments they encounter within secondary organs to facilitate metastatic colonization. Our study elucidated that disseminated osteosarcoma cells survive within the lung by co-opting and amplifying the lung's endogenous wound healing response program. More broadly, our results support a model wherein mesenchymal-epithelial cooperation is a key driver of lung metastasis. Osteosarcoma, a pediatric mesenchymal tumor, undergoes lung epithelial induced fibrotic activation while also transforming normal lung epithelial cells towards a fibrosis promoting phenotype. Conversely, adult epithelial carcinomas activate fibrotic signaling in normal lung mesenchymal fibroblasts. Our data implicates fibrosis and abnormal wound healing as key drivers of lung metastasis across multiple tumor types that can be targeted therapeutically to disrupt metastasis progression.

Host-derived growth factors drive ERK phosphorylation and MCL1 expression to promote osteosarcoma cell survival during metastatic lung colonization.

PURPOSE: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. METHODS: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. RESULTS: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. CONCLUSION: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.

Osteosarcoma Multi-Omics Landscape and Subtypes.

Osteosarcoma (OS) is the most common primary bone malignancy that exhibits remarkable histologic diversity and genetic heterogeneity. The complex nature of osteosarcoma has confounded precise molecular categorization, prognosis, and prediction for this disease. In this study, we performed a comprehensive multiplatform analysis on 86 osteosarcoma tumors, including somatic copy-number alteration, gene expression and methylation, and identified three molecularly distinct and clinically relevant subtypes of osteosarcoma. The subgrouping criteria was validated on another cohort of osteosarcoma tumors. Previously unappreciated osteosarcoma-type-specific changes in specific genes' copy number, expression and methylation were revealed based on the subgrouping. The subgrouping and novel gene signatures provide insights into refining osteosarcoma therapy and relationships to other types of cancer.

Post-zygotic rescue of meiotic errors causes brain mosaicism and focal epilepsy.

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development.

Oncolytic virus-driven immune remodeling revealed in mouse medulloblastomas at single cell resolution.

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma.

Structurally Complex Osteosarcoma Genomes Exhibit Limited Heterogeneity within Individual Tumors and across Evolutionary Time.

Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNA) are the genetic drivers of disease. Models around genomic instability conflict-it is unclear whether osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single-cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single-cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single-cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little subclonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic timepoints (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. Significance: Chromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications for diagnosis, biomarker assessment, mechanisms of treatment resistance, and represents a conceptual advance in our understanding of intratumoral heterogeneity and tumor evolution.

Osteosarcoma tumors maintain intra-tumoral transcriptional heterogeneity during bone and lung colonization.

BACKGROUND: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. RESULTS: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. CONCLUSIONS: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma.

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ T cells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

Nuclear receptor Nr5a2 promotes diverse connective tissue fates in the jaw.

Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.

Long-Wavelength Fluctuations in Quasi-2D Supercooled Liquids.

We use molecular simulation to characterize the dynamics of supercooled liquids confined in quasi-2D slit geometries. Similar to bulk supercooled liquids, the confined systems exhibit subdiffusive dynamics on intermediate time scales arising from particle localization inside their neighbor cages, followed by an eventual crossover to diffusive behavior as cage rearrangement occurs. The quasi-2D confined liquids also exhibit signatures of long-wavelength fluctuations (LWFs) in the lateral directions parallel to the confining walls, reminiscent of the collective displacements observed in 2D but not 3D systems. The magnitude of the LWFs increases with the lateral dimensions of systems with the same particle volume fraction and confinement length scale, consistent with the logarithmic scaling predicted for 2D Mermin-Wagner fluctuations. The amplitude of the fluctuations is a nonmonotonic function of the confinement length scale because of a competition between caging and strengthening LWFs upon approaching the 2D limit. Our findings suggest that LWFs may play an important role in understanding the behavior of confined supercooled liquids due to their prevalence over a surprisingly broad range of particle densities and confinement length scales.

Redundant Signaling as the Predominant Mechanism for Resistance to Antibodies Targeting the Type-I Insulin-Like Growth Factor Receptor in Cells Derived from Childhood Sarcoma.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.

Molecularly Defined Subsets of Ewing Sarcoma Tumors Differ in Their Responses to IGF1R and WEE1 Inhibition.

PURPOSE: Targeted cancer therapeutics have not significantly benefited patients with Ewing sarcoma with metastatic or relapsed disease. Understanding the molecular underpinnings of drug resistance can lead to biomarker-driven treatment selection. EXPERIMENTAL DESIGN: Receptor tyrosine kinase (RTK) pathway activation was analyzed in tumor cells derived from a panel of Ewing sarcoma tumors, including primary and metastatic tumors from the same patient. Phospho-RTK arrays, Western blots, and IHC were used. Protein localization and the levels of key markers were determined using immunofluorescence. DNA damage tolerance was measured through PCNA ubiquitination levels and the DNA fiber assay. Effects of pharmacologic inhibition were assessed in vitro and key results validated in vivo using patient-derived xenografts. RESULTS: Ewing sarcoma tumors fell into two groups. In one, IGF1R was predominantly nuclear (nIGF1R), DNA damage tolerance pathway was upregulated, and cells had low replication stress and RRM2B levels and high levels of WEE1 and RAD21. These tumors were relatively insensitive to IGF1R inhibition. The second group had high replication stress and RRM2B, low levels of WEE1 and RAD21, membrane-associated IGF1R (mIGF1R) signaling, and sensitivity to IGF1R or WEE1-targeted inhibitors. Moreover, the matched primary and metastatic tumors differed in IGF1R localization, levels of replication stress, and inhibitor sensitivity. In all instances, combined IGF1R and WEE1 inhibition led to tumor regression. CONCLUSIONS: IGF1R signaling mechanisms and replication stress levels can vary among Ewing sarcoma tumors (including in the same patient), influencing the effects of IGF1R and WEE1 treatment. These findings make the case for using biopsy-derived predictive biomarkers at multiple stages of Ewing sarcoma disease management.

EGFR internal tandem duplications in fusion-negative congenital and neonatal spindle cell tumors.

Next-generation sequencing (NGS) assays can sensitively detect somatic variation, and increasingly can enable the identification of complex structural rearrangements. A subset of infantile spindle cell sarcomas, particularly congenital mesoblastic nephromas with classic or mixed histology, have structural rearrangement in the form of internal tandem duplications (ITD) involving EGFR. We performed prospective analysis to identify EGFR ITD through clinical or research studies, as well as retrospective analysis to quantify the frequency of EGFR ITD in pediatric sarcomas. Within our institution, three tumors with EGFR ITD were prospectively identified, all occurring in patients less than 1 year of age at diagnosis, including two renal tumors and one mediastinal soft tissue tumor. These three cases exhibited both cellular and mixed cellular and classic histology. All patients had no evidence of disease progression off therapy, despite incomplete resection. To extend our analysis and quantify the frequency of EGFR ITD in pediatric sarcomas, we retrospectively analyzed a cohort of tumors (n = 90) that were previously negative for clinical RT-PCR-based fusion testing. We identified EGFR ITD in three analyzed cases, all in patients less than 1 year of age (n = 18; 3/18, 17%). Here we expand the spectrum of tumors with EGFR ITD to congenital soft tissue tumors and report an unusual example of an EGFR ITD in a tumor with cellular congenital mesoblastic nephroma histology. We also highlight the importance of appropriate test selection and bioinformatic analysis for identification of this genomic alteration that is unexpectedly common in congenital and infantile spindle cell tumors.

Osteosarcoma.

Osteosarcoma is the most common primary malignant tumour of the bone. Osteosarcoma incidence is bimodal, peaking at 18 and 60 years of age, and is slightly more common in males. The key pathophysiological mechanism involves several possible genetic drivers of disease linked to bone formation, causing malignant progression and metastasis. While there have been significant improvements in the outcome of patients with localized disease, with event-free survival outcomes exceeding 60%, in patients with metastatic disease, event-free survival outcomes remain poor at less than 30%. The suspicion of osteosarcoma based on radiographs still requires pathological evaluation of a bone biopsy specimen for definitive diagnosis and CT imaging of the chest should be performed to identify lung nodules. So far, population-based screening and surveillance strategies have not been implemented due to the rarity of osteosarcoma and the lack of reliable markers. Current screening focuses only on groups at high risk such as patients with genetic cancer predisposition syndromes. Management of osteosarcoma requires a multidisciplinary team of paediatric and medical oncologists, orthopaedic and general surgeons, pathologists, radiologists and specialist nurses. Survivors of osteosarcoma require specialized medical follow-up, as curative treatment consisting of chemotherapy and surgery has long-term adverse effects, which also affect the quality of life of patients. The development of osteosarcoma model systems and related research as well as the evaluation of new treatment approaches are ongoing to improve disease outcomes, especially for patients with metastases.

An international working group consensus report for the prioritization of molecular biomarkers for Ewing sarcoma.

The advent of dose intensified interval compressed therapy has improved event-free survival for patients with localized Ewing sarcoma (EwS) to 78% at 5 years. However, nearly a quarter of patients with localized tumors and 60-80% of patients with metastatic tumors suffer relapse and die of disease. In addition, those who survive are often left with debilitating late effects. Clinical features aside from stage have proven inadequate to meaningfully classify patients for risk-stratified therapy. Therefore, there is a critical need to develop approaches to risk stratify patients with EwS based on molecular features. Over the past decade, new technology has enabled the study of multiple molecular biomarkers in EwS. Preliminary evidence requiring validation supports copy number changes, and loss of function mutations in tumor suppressor genes as biomarkers of outcome in EwS. Initial studies of circulating tumor DNA demonstrated that diagnostic ctDNA burden and ctDNA clearance during induction are also associated with outcome. In addition, fusion partner should be a pre-requisite for enrollment on EwS clinical trials, and the fusion type and structure require further study to determine prognostic impact. These emerging biomarkers represent a new horizon in our understanding of disease risk and will enable future efforts to develop risk-adapted treatment.

Lurbinectedin Inhibits the EWS-WT1 Transcription Factor in Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric sarcoma with poor overall survival. This tumor is absolutely dependent on the continued expression and activity of its defining molecular lesion, the EWS-WT1 transcription factor. Unfortunately, the therapeutic targeting of transcription factors is challenging, and there is a critical need to identify compounds that inhibit EWS-WT1. Here we show that the compound lurbinectedin inhibits EWS-WT1 by redistributing the protein within the nucleus to the nucleolus. This nucleolar redistribution interferes with the activity of EWS-WT1 to reverse the expression of over 70% of the transcriptome. In addition, the compound blocks the expression of the EWS-WT1 fusion protein to inhibit cell proliferation at the lowest GI50 ever reported for this compound in any cell type. The effects occur at concentrations that are easily achievable in the clinic and translate to the in vivo setting to cause tumor regressions in multiple mice in a xenograft and PDX model of DSRCT. Importantly, this mechanism of nucleolar redistribution is also seen with wild-type EWSR1 and the related fusion protein EWS-FLI1. This provides evidence for a "class effect" for the more than 18 tumors driven by EWSR1 fusion proteins. More importantly, the data establish lurbinectedin as a promising clinical candidate for DSRCT.

Development of a multi faceted platform containing a tetrazine, fluorophore and chelator: synthesis, characterization, radiolabeling, and immuno-SPECT imaging.

BACKGROUND: Combining optical (fluorescence) imaging with nuclear imaging has the potential to offer a powerful tool in personal health care, where nuclear imaging offers in vivo functional whole-body visualization, and the fluorescence modality may be used for image-guided tumor resection. Varying chemical strategies have been exploited to fuse both modalities into one molecular entity. When radiometals are employed in nuclear imaging, a chelator is typically inserted into the molecule to facilitate radiolabeling; the availability of the chelator further expands the potential use of these platforms for targeted radionuclide therapy if a therapeutic radiometal is employed. Herein, a novel mixed modality scaffold which contains a tetrazine (Tz)--for biomolecule conjugation, fluorophore-for optical imaging, and chelator-for radiometal incorporation, in one construct is presented. The novel platform was characterized for its fluorescence properties, radiolabeled with single-photon emission computed tomography (SPECT) isotope indium-111 (111In3+) and therapeutic alpha emitter actinium-225 (225Ac3+). Both radiolabels were conjugated in vitro to trans-cyclooctene (TCO)-modified trastuzumab; biodistribution and immuno-SPECT imaging of the former conjugate was assessed. RESULTS: Key to the success of the platform synthesis was incorporation of a 4,4'-dicyano-BODIPY fluorophore. The route gives access to an advanced intermediate where final chelator-incorporated compounds can be easily accessed in one step prior to radiolabeling or biomolecule conjugation. The DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugate was prepared, displayed good fluorescence properties, and was successfully radiolabeled with 111In & 225Ac in high radiochemical yield. Both complexes were then separately conjugated in vitro to TCO modified trastuzumab through an inverse electron demand Diels-Alder (IEDDA) reaction with the Tz. Pilot small animal in vivo immuno-SPECT imaging with [111In]In-DO3A-BODIPY-Tz-TCO-trastuzumab was also conducted and exhibited high tumor uptake (21.2 ± 5.6%ID/g 6 days post-injection) with low uptake in non-target tissues. CONCLUSIONS: The novel platform shows promise as a multi-modal probe for theranostic applications. In particular, access to an advanced synthetic intermediate where tailored chelators can be incorporated in the last step of synthesis expands the potential use of the scaffold to other radiometals. Future studies including validation of ex vivo fluorescence imaging and exploiting the pre-targeting approach available through the IEDDA reaction are warranted.