SUMMARY Sonic hedgehog (Shh), a soluble ligand overexpres sed by neoplastic cells in pancreatic ductal adenocarcinoma (PDAC), drives formation of a fibroblast-rich desmoplastic stroma. To better understand its role in malignant progression, we deleted Shh in a well-defined mouse model of PDAC. As predicted, Shh-deficient tumors had reduced stromal content. Surprisingly, such tumors were more aggressive and exhibited undifferentiated histology, increased vascularity, and heightened proliferation – features that were fully recapitulated in control mice treated with a Smoothened inhibitor. Furthermore, administration of VEGFR blocking antibody selectively improved survival of Shh-deficient tumors, indicating that Hedgehog-driven stroma suppresses tumor growth in part by restraining tumor angiogenesis. Together, these data demonstrate that some components of the tumor stroma can act to restrain tumor growth.

The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the tumor extent is desired clinically and surgically. Here we show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal loco-regional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed here SERRS-nanostars. The SERRS-nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, SERRS-nanostars enabled accurate detection of macroscopic malignant lesions as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 femtomolar limit of detection under in vivo Raman imaging conditions) of SERRS-nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS-nanostars a promising imaging agent for more precise cancer imaging and resection.

Conditional gene inactivation using the Cre/loxP system has lead to significant advances in our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature, that Cre transgenic mice may themselves have a phenotype. In the following study we describe the bone phenotype of a commonly used Cre transgenic mouse line to study osteoblasts, the Osx-GFP::Cre (Osx-Cre) mice. Cortical and trabecular bone parameters were determined in the femurs of Osx-Cre mice at 6 and 12 weeks of age by microtomography (μCT). At 6 weeks of age, Osx-Cre mice had reduced body weight by 22% (P < 0.0001) and delayed cortical bone expansion and accrual, characterized by decreases in periosteal circumference by 7% (P < 0.05) and cortical thickness by 11% (P < 0.01), compared to wild type controls. Importantly, the cortical bone phenotype of the skeletally immature Osx-Cre mice at 6 weeks of age could be accounted for by their low body weight. The delayed weight gain and cortical growth of Osx-Cre mice was overcome by 12 weeks of age, with no differences observed between Osx-Cre and wild type controls. In conclusion, Osx-Cre expressing mice display a delayed growth phenotype in the absence of doxycycline treatment, evidenced by decreased cortical bone expansion and accrual at 6 weeks of age, as an indirect result of decreased body weight. While this delay in growth is overcome by adulthood at 12 weeks of age, caution together with appropriate data analysis must be considered when assessing the experimental data from skeletally immature Cre/loxP knockout mice generated using the Osx-Cre mouse line to avoid misinterpretation.

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.

The process of advanced glycation leads to irreversible cross-linking of long-lived proteins [1]. This process is accentuated in diabetes and has been postulated to contribute to the development of a range of diabetic complications including nephropathy [2], retinopathy [3] and neuropathy [4]. Aminoguanidine (AG) has previously been shown to retard the development of advanced glycation end products (AGEs) in various tissues in streptozotocin diabetes [5]. In those studies, AG was shown to decrease mesangial expansion and albuminuria, both markers of the

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During lactation, the large transfer of calcium from the mother to the milk is primarily sourced from the maternal skeleton. To determine whether the calcitonin receptor (CTR) plays a physiological role to protect the skeleton from excessive resorption during lactation, we assessed the maternal skeleton of global CTR knockout (CTRKO) and littermate control mice at the end of lactation (postnatal day 21). Micro-computed tomography analyses showed no effect on trabecular or cortical bone in the distal femur and L1 vertebra of maternal global CTR deletion at the end of lactation in global CTRKO mice compared with that in control mice. Bone resorption, as assessed by osteoclast number and activity at the end of lactation, was unaffected by maternal CTR deletion. Cathepsin K, carbonic anhydrase 2, matrix metalloproteinase 13, and receptor activator of nuclear factor-κB ligand mRNA levels, however, were markedly elevated by 3- to 6.5-fold in whole bone of lactating global CTRKO females. Because these genes have been shown to be up-regulated in osteocytes during lactation when osteocytes resorb their surrounding bone matrix, together with their reported expression of the CTR, we determined the osteocyte lacunar area in cortical bone. After lactation, the top 20% of osteocyte lacunar area in global CTRKO mice was 10% larger than the top 20% in control mice. These data are consistent with an increased osteocytic osteolysis in global CTRKO mice during lactation, which is further supported by the increased serum calcium observed in global CTRKO mice after lactation. These results provide evidence for a physiological role for the CTR to protect the maternal skeleton during lactation by a direct action on osteocytes to inhibit osteolysis.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer that is broadly chemoresistant, due in part to biophysical properties of tumor stroma, which serves as a barrier to drug delivery for most classical chemotherapeutic drugs. The goal of this work is to evaluate the preclinical efficacy and mechanisms of PTC596, a novel agent with potent anticancer properties in vitro and desirable pharmacologic properties in vivo. Experimental Design: We assessed the pharmacology, mechanism, and preclinical efficacy of PTC596 in combination with standards of care, using multiple preclinical models of PDA. Results: We found that PTC596 has pharmacologic properties that overcome the barrier to drug delivery in PDA, including a long circulating half-life, lack of P-glycoprotein substrate activity, and high systemic tolerability. We also found that PTC596 combined synergistically with standard clinical regimens to improve efficacy in multiple model systems, including the chemoresistant genetically engineered "KPC" model of PDA. Through mechanistic studies, we learned that PTC596 functions as a direct microtubule polymerization inhibitor, yet a prior clinical trial found that it lacks peripheral neurotoxicity, in contrast to other such agents. Strikingly, we found that PTC596 synergized with the standard clinical backbone regimen gemcitabine/ nab-paclitaxel, yielding potent, durable regressions in a PDX model. Moreover, similar efficacy was achieved in combination with nab-paclitaxel alone, highlighting a specific synergistic interaction between two different microtubule-targeted agents in the setting of pancreatic ductal adenocarcinoma. Conclusions: These data demonstrate clear rationale for the development of PTC596 in combination with standard-ofcare chemotherapy for PDA.