Natalie J. Serkova

Multispectral imaging is a cornerstone of chemical imaging, with bioluminescence, fluorescence, and phosphorescence imaging underpin much of preclinical and analytical chemical measurements. However, conventional implementations are typically proprietary, costly, and resolve spectral content from the reflectance of a broadband source, without control over the excitation spectrum. This architecture cannot isolate excitation-dependent photophysical processes. Spectral discrimination through sequentially resolved narrowband excitation, detected on a single broadband-sensitive camera, instead enables excitation-dependent fluorescence, phosphorescence, and reflectance measurements not available to such broadband approaches. We describe AURORA-MSI, an open-source multispectral platform that inverts this arrangement with fourteen narrowband LEDs spanning 367-940 nm, a 120-element annular illumination ring with eight independently addressable azimuthal sectors, and a thermoelectrically cooled monochrome CMOS camera. Radiometric calibration, spatial uniformity mapping, and camera noise characterization establish the quantitative measurement foundation. In bioluminescence imaging, AURORA-MSI localized sources in a calibrated tissue-mimicking mouse phantom comparably to a commercial Revvity IVIS Spectrum, detected luciferase-expressing HSJD-GBM1-001 glioblastoma cells across a dilution series, with reduced replicate consistency at the lowest densities, and mapped substrate-free fungal-pathway emission in an intact bioluminescent Petunia hybrida , none requiring photon-counting instrumentation. Time-resolved phosphorescence decay imaging of six inorganic phosphors over 33 minutes found tri-exponential models adequate at early times, while distributed-lifetime models were preferred at extended timescales. Multispectral image-texture features extracted across all fourteen excitation bands discriminated ten pharmaceutical and household powders spanning distinct chemical compositions and, in two cases, distinct formulations of the same compound. Generality beyond these regimes was established through dual-excitation fluorescence fingerprinting of fifteen mineral specimens and wavelength-selective plant-tissue imaging exploiting UV and NIR penetration-depth differences. Excitation-side spectral encoding enables photophysical measurements that detection-side systems with uncontrolled broadband illumination cannot isolate, and the cooled-camera architecture supports weak-signal modalities without photon-counting instrumentation.

Estrogen receptor-positive breast cancer represents a significant proportion of breast cancer brain metastasis but remains understudied. Here we show that FGFR1-amplification, a well-established driver of estrogen receptor-positive breast cancer endocrine resistance, promotes estrogen receptor-positive breast cancer brain metastatic colonization in young and aged female mice, through both canonical FGF2/FGFR1 signaling and non-canonical NCAM1/FGFR1 interactions. Astrocytic FGF2-mediated paracrine activation of FGFR1 promotes breast cancer brain metastasis in estrogen-treated young mice, but FGF2 levels and signaling decrease in the brain with aging and estrogen-depletion. Neuronal and astrocytic NCAM1, which remain unchanged in young and aged brains, promote adhesion to neurons, migration, and growth of estrogen receptor-positive cells, suggesting that interactions with astrocytes and neurons facilitate early estrogen receptor-positive breast cancer brain metastasis colonization through FGFR1. Importantly, FDA-approved FGFR inhibitors effectively block early colonization but not late-stage brain metastases, suggesting prevention of FGFR1+ brain metastases as a window of opportunity for FGFR1 inhibitors.

MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening, we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover, we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program, as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets, reduces ribosome biogenesis, and impairs protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore, combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo, leading to more pronounced tumor growth suppression. Overall, our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB.

Confirmation of ATRT growth dependency on SIRT2 with rescue vector. A. Western Blot demonstrates shRNA mediated knockdown in BT16(MYC) cells and restoring SIRT2 level with ORF rescue vector for SIRT2. B. Membranes for Western Blot assay. C. Colony formation assay with colonies images and quantification. Genetic knockdown of SIRT2 decreases SIRT2 mRNA in BT16(MYC). Rescue vector for SIRT2 confirms growth dependency on SIRT2 in ATRT. ( **p<0.01). D. Real time cell assay with cell growth images and quantification. SIRT2 depletion inhibits BT16(MYC) proliferation as compared to shNULL ( *p<0.05). Rescue vector confirms growth dependency on SIRT2 in ATRT.

SIRT2 is essential for ATRT growth. A. Genetic knockdown SIRT2 decreases SIRT2 mRNA in BT16(MYC) and MAF737(TYR) cells as measured by RT qPCR assay ( **p<0.01). B. Western Blot of SIRT2 demonstrates shRNA mediated knockdown in BT16(MYC) and MAF737(TYR) cells. C. Membranes for Western Blot assay (Figure S2.B). D. Real time cell assay with cell growth images and quantification. SIRT2 depletion inhibits BT16(MYC) and MAF737(TYR) ATRT proliferation as compared to shNULL(*p<0.05).

An atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor driven by the loss of SMARCB1, which results in epigenetic dysregulation of the genome. SMARCB1 loss affects lineage commitment and differentiation by controlling gene expression. We hypothesized that additional epigenetic factors cooperate with SMARCB1 loss to control cell self-renewal and drive ATRT. We performed an unbiased epigenome-targeted screen to identify genes that cooperate with SMARCB1 and identified SIRT2 as a key regulator. Using in vitro pluripotency assays combined with in vivo single-cell RNA transcriptomics, we examined the impact of SIRT2 on differentiation of ATRT cells. We used a series of orthotopic murine models treated with SIRT2 inhibitors to examine the impact on survival and clinical applicability. We found that ATRT cells are highly dependent on SIRT2 for survival. Genetic or chemical inhibition led to decreased cell self-renewal and induction of differentiation in tumor spheres and in vivo models. We found that SIRT2 inhibition can restore gene expression programs lost because of SMARCB1 loss and reverse the differentiation block in ATRT in vivo. Finally, we showed the in vivo efficacy of a clinically relevant inhibitor demonstrating SIRT2 inhibition as a potential therapeutic strategy. We concluded that SIRT2 is a critical dependency in SMARCB1-deficient ATRT cells and acts by controlling the pluripotency–differentiation switch. Thus, SIRT2 inhibition is a promising therapeutic approach that warrants further investigation and clinical development.Implications:SIRT2 inhibition is a molecular vulnerability in SMARCB1-deleted tumors.

All hits in MAF737 ATRT from shRNA screen with fold change<0.8; p<0.05. One of the top hits is SIRT2 (fold change = 0.309, p<0.05).

SIRT2 inhibition suppressed BT16(MYC) ATRT growth in vivo. A. Bioluminescence images of orthotopic ATRT tumors treated with Vehicle (Control) or TM. B. Survival analysis of Vehicle vs TM treated animals (median 19 days vs 33 days, log rank test p<0.05, n=4). C. Immunohistochemical (IHC) stains of cerebellar tumors from BT16(MYC) injected mice are shown (40x magnification). FOXM1 accumulation in mice tumors 4 weeks TM treatment. D. Quantification of Image C. Plots show values from quantification of representative images.TM treatment decreased oncogenic marker FOXM1 (p<0.05). E. Ki67 accumulation in mice tumors 4 weeks TM treatment (40x). F. Quantification of Image E. Plots show values from quantification representative images. Stain accumulation from 5 fields of each slide were analyzed using ImageJ program.TM treatment decreased oncogenic marker KI67 (* p<0.05). G. MEF2A accumulation in mice tumors 4 weeks TM treatment (40x). H. Quantification of Image G (40x magnification). Plots show values from quantification representative images. Stain accumulation from 5 fields of each slide were analyzed using ImageJ program. TM treatment increased accumulation neuronal differentiation marker MEF2A (p<0.05).

Colony Formation Assay and SIRT2 expression in ATRT subgroups. A. CFA for hits genes in MAF737 ATRT from shRNA screen. Representative images of colony formations. B. Quantification of CFA. SIRT2 depletion results in a significant loss of ATRT cell viability (*p <0.001). C. Expression of SIRT2 RNA in patient cohorts in all ATRT subgroups as compared to normal cerebellum. Data from Children's Hospital Colorado.

H&E stained sections showed a high-grade neoplasm consisting of mildly pleomorphic tumor cells with large vesicular chromatin, occasional prominent nucleoli (arrowheads), increased mitoses and zonal necrosis (asterisk). Scattered cells showed rhabdoid features with cytoplasmic eosinophilic inclusions (arrow) and eccentrically placed nuclei.