Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Cleft Palate and Aglossia Result From Perturbations in Wnt and Hedgehog Signaling.

OBJECTIVE: The objective of this study was to explore the molecular basis for cleft secondary palate and arrested tongue development caused by the loss of the intraflagellar transport protein, Kif3a. DESIGN: Kif3a mutant embryos and their littermate controls were analyzed for defects in facial development at multiple stages of embryonic development. Histology was employed to understand the effects of Kif3a deletion on palate and tongue development. Various transgenic reporter strains were used to understand how deletion of Kif3a affected Hedgehog and Wnt signaling. Immunostaining for structural elements of the tongue and for components of the Wnt pathway were performed. BrdU activity analyses were carried out to examine how the loss of Kif3a affected cell proliferation and led to palate and tongue malformations. RESULTS: Kif3a deletion causes cranial neural crest cells to become unresponsive to Hedgehog signals and hyper-responsive to Wnt signals. This aberrant molecular signaling causes abnormally high cell proliferation, but paradoxically outgrowths of the tongue and the palatal processes are reduced. The basis for this enigmatic effect can be traced back to a disruption in epithelial/mesenchymal signaling that governs facial development. CONCLUSION: The primary cilium is a cell surface organelle that integrates Hh and Wnt signaling, and disruptions in the function of the primary cilium cause one of the most common-of the rarest-craniofacial birth defects observed in humans. The shared molecular basis for these dysmorphologies is an abnormally high Wnt signal simultaneous with an abnormally low Hedgehog signal. These pathways are integrated in the primary cilium.

Hormone sensitive lipase ablation promotes bone regeneration.

There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial µCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

Towards Quantification of Functional Breast Images Using Dedicated SPECT With Non-Traditional Acquisition Trajectories.

Quantification of radiotracer uptake in breast lesions can provide valuable information to physicians in deciding patient care or determining treatment efficacy. Physical processes (e.g., scatter, attenuation), detector/collimator characteristics, sampling and acquisition trajectories, and reconstruction artifacts contribute to an incorrect measurement of absolute tracer activity and distribution. For these experiments, a cylinder with three syringes of varying radioactivity concentration, and a fillable 800 mL breast with two lesion phantoms containing aqueous (99m)Tc pertechnetate were imaged using the SPECT sub-system of the dual-modality SPECT-CT dedicated breast scanner. SPECT images were collected using a compact CZT camera with various 3D acquisitions including vertical axis of rotation, 30° tilted, and complex sinusoidal trajectories. Different energy windows around the photopeak were quantitatively compared, along with appropriate scatter energy windows, to determine the best quantification accuracy after attenuation and dual-window scatter correction. Measured activity concentrations in the reconstructed images for syringes with greater than 10 µCi /mL corresponded to within 10% of the actual dose calibrator measured activity concentration for ±4% and ±8% photopeak energy windows. The same energy windows yielded lesion quantification results within 10% in the breast phantom as well. Results for the more complete complex sinsusoidal trajectory are similar to the simple vertical axis acquisition, and additionally allows both anterior chest wall sampling, no image distortion, and reasonably accurate quantification.

STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets.

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/ß sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.

Generation of bone marrow chimeras using X-ray irradiation: comparison to cesium irradiation and use in immunotherapy.

Bone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities-including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models-specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types.

Aberrantly elevated Wnt signaling is responsible for cementum overgrowth and dental ankylosis.

Vertebrate teeth are attached to the jawbones using a variety of methods but in mammals, a fibrous connection is the norm. This fibrous periodontal ligament (PDL) allows teeth to move in the jawbones in response to natural eruptive forces, mastication, and orthodontic tooth movement. In some disease states the PDL either calcifies or is replaced by a mineralized tissue and the result is ankylosis, where the tooth is fused to the alveolar bone. To understand how the PDL maintains this fibrous state, we examined a strain of mice in which tooth movement is arrested. DaßcatOt mice express a stabilized form of ß-catenin in DMP1-positive alveolar bone osteocytes and cementocytes, which results in elevated Wnt signaling throughout the periodontium. As a consequence, there is an accrual of massive amounts of cellular cementum and alveolar bone, the PDL itself calcifies and teeth become ankylosed. These data suggest that to maintain its fibrous nature, Wnt signaling must normally be repressed in the PDL space.

CB-6644 Is a Selective Inhibitor of the RUVBL1/2 Complex with Anticancer Activity.

RUVBL1 and RUVBL2 are ATPases associated with diverse cellular activities (AAAs) that form a complex involved in a variety of cellular processes, including chromatin remodeling and regulation of gene expression. RUVBLs have a strong link to oncogenesis, where overexpression is correlated with tumor growth and poor prognosis in several cancer types. CB-6644, an allosteric small-molecule inhibitor of the ATPase activity of the RUVBL1/2 complex, interacts specifically with RUVBL1/2 in cancer cells, leading to cell death. Importantly, drug-acquired-resistant cell clones have amino acid mutations in either RUVBL1 or RUVBL2, suggesting that cell killing is an on-target consequence of RUVBL1/2 engagement. In xenograft models of acute myeloid leukemia and multiple myeloma, CB-6644 significantly reduced tumor growth without obvious toxicity. This work demonstrates the therapeutic potential of targeting RUVBLs in the treatment of cancer and establishes a chemical entity for probing the many facets of RUVBL biology.

Initial In Vivo Quantification of Tc-99m Sestamibi Uptake as a Function of Tissue Type in Healthy Breasts Using Dedicated Breast SPECT-CT.

A pilot study is underway to quantify in vivo the uptake and distribution of Tc-99m Sestamibi in subjects without previous history of breast cancer using a dedicated SPECT-CT breast imaging system. Subjects undergoing diagnostic parathyroid imaging studies were consented and imaged as part of this IRB-approved breast imaging study. For each of the seven subjects, one randomly selected breast was imaged prone-pendant using the dedicated, compact breast SPECT-CT system underneath the shielded patient support. Iteratively reconstructed and attenuation and/or scatter corrected images were coregistered; CT images were segmented into glandular and fatty tissue by three different methods; the average concentration of Sestamibi was determined from the SPECT data using the CT-based segmentation and previously established quantification techniques. Very minor differences between the segmentation methods were observed, and the results indicate an average image-based in vivo Sestamibi concentration of 0.10 ± 0.16 µCi/mL with no preferential uptake by glandular or fatty tissues.

Is SPECT or CT Based Attenuation Correction More Quantitatively Accurate for Dedicated Breast SPECT Acquired with Non-Traditional Trajectories?

Attenuation correction is necessary for SPECT quantification. There are a variety of methods to create attenuation maps. For dedicated breast SPECT imaging, it is unclear if either SPECT- or CT-based attenuation map would provide the most accurate quantification and whether or not segmenting the different tissue types will have an effect on the qunatification. For these experiments, 99mTc diluted in methanol and water was filled into geometric and anthropomorphic breast phantoms and was imaged with a dedicated dual-modality SPECT-CT scanner. SPECT images were collected using a compact CZT camera with various 3D acquisitions including vertical and 30° tilted parallel beam, and complex sinusoidal trajectories. CT images were acquired using a quasi-monochromatic x-ray source and CsI(T1) flat panel digital detector in a half-cone beam geometry. Measured scatter correction for SPECT and CT were implemented. To compare photon attenuation correction in the reconstructed SPECT images, various volumetric attenuation matrices were derived from 1) uniform SPECT, 2) uniform CT, and 3) segmented CT, populated with different attenuation coefficient values. Comparisons between attenuation masks using phantoms consisting of materials with different attenuation values show that at 140 keV the differences in the attenuation between materials do not affect the quantification as much as the size and alignment of the attenuation map. The CT-based attenuation maps give quantitative values 30% below the actual value, but are consistent. While the SPECT-based attenuation maps can provide within 10% accurate quantitative values, but are less consistent.