Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.

CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.

Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines.

Recent progress has shown that using wavelengths between 1,000 and 2,000 nm, referred to as the shortwave-infrared or near-infrared (NIR)-II range, can enable high-resolution in vivo imaging at depths not possible with conventional optical wavelengths. However, few bioconjugatable probes of the type that have proven invaluable for multiplexed imaging in the visible and NIR range are available for imaging these wavelengths. Using rational design, we have generated persulfonated indocyanine dyes with absorbance maxima at 872 and 1,072 nm through catechol-ring and aryl-ring fusion, respectively, onto the nonamethine scaffold. Multiplexed two-color and three-color in vivo imaging using monoclonal antibody and dextran conjugates in several tumor models illustrate the benefits of concurrent labeling of the tumor and healthy surrounding tissue and lymphatics. These efforts are enabled by complementary advances in a custom-built NIR/shortwave-infrared imaging setup and software package for multicolor real-time imaging.

Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging.

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4'-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1, with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.

3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) Positron Emission Tomography as an In Vivo Biomarker of inhibition of CDK 4/6-Rb pathway by Palbociclib in a patient derived bladder tumor.

BACKGROUND: Several new generation CDK4/6 inhibitors have been developed and approved for breast cancer therapy in combination with endocrine therapeutics. Application of these inhibitors either alone or in combination in other solid tumors has been proposed, but no imaging biomarkers of response have been reported in non-breast cancer animal models. The purpose of this study was to evaluate 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) Positron Emission Tomography (PET) as in vivo biomarker of response to palbociclib in a non-breast cancer model. METHODS: Twenty-four NSG mice bearing patient derived xenografts (PDX) of a well-characterized bladder tumor were randomized into 4 treatment groups: vehicle (n = 6); palbociclib (n = 6); temozolomide (n = 6); and palbociclib plus temozolomide (n = 6) and treated with two cycles of therapy or vehicle. Tumor uptake of [18F]FLT was determined by micro-PET/CT at baseline, 3 days, and 9 days post initiation of therapy. Following the second cycle of therapy, the mice were maintained until their tumors reached a size requiring humane termination. RESULTS: [18F]FLT uptake decreased significantly in the palbociclib and combination arms (p = 0.0423 and 0.0106 respectively at day 3 and 0.0012 and 0.0031 at day 9) with stable tumor volume. In the temozolomide arm [18F]FLT uptake increased with day 9 uptake significantly different than baseline (p = 0.0418) and progressive tumor growth was observed during the treatment phase. All groups exhibited progressive disease after day 22, 10 days following cessation of therapy. CONCLUSION: Significant decreases in [18F]FLT uptake as early as three days post initiation of therapy with palbociclib, alone or in combination with temozolomide, in this bladder cancer model correlates with an absence of tumor growth during therapy that persists until day 18 for the palbociclib group and day 22 for the combination group (6 days and 10 days) following cessation of therapy. These results support early modulation of [18F]FLT as an in vivo biomarker predictive of palbociclib therapy response in a non-breast cancer model.

Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting.

Conjugates of small molecules and antibodies are broadly employed diagnostic and therapeutic agents. Appending a small molecule to an antibody often significantly impacts the properties of the resulting conjugate. Here, we detail a systematic study investigating the effect of various functional groups on the properties of antibody-fluorophore conjugates. This was done through the preparation and analysis of a series of masked heptamethine cyanines (CyMasks)-bearing amides with varied functional groups. These were designed to exhibit a broad range of physical properties, and include hydrophobic (-NMe2), pegylated (NH-PEG-8 or NH-PEG-24), cationic (NH-(CH2)2NMe3+), anionic (NH-(CH2)2SO3-), and zwitterionic (N-(CH2)2NMe3+)-(CH2)3SO3-) variants. The CyMask series was appended to monoclonal antibodies (mAbs) and analyzed for the effects on tumor targeting, clearance, and non-specific organ uptake. Among the series, zwitterionic and pegylated dye conjugates had the highest tumor-to-background ratio (TBR) and a low liver-to-background ratio. By contrast, the cationic and zwitterionic probes had high tumor signal and high TBR, although the latter also exhibited an elevated liver-to-background ratio (LBR). Overall, these studies provide a strategy to test the functional group effects and suggest that zwitterionic substituents possess an optimal combination of high tumor signal, TBR, and low LBR. These results suggest an appealing strategy to mask hydrophobic payloads, with the potential to improve the properties of bioconjugates in vivo.

Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry.

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitatively assess the cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits a dramatically increased cellular signal due to an improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ∼50× brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or nonhindered disulfides, an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

A spontaneously metastatic model of bladder cancer: imaging characterization.

BACKGROUND: Spontaneously metastatic xenograft models of cancer are infrequent and the few that exist are resource intensive. In xenografts, caliper measurements can be used to determine primary tumor burden and response to therapy but in metastatic disease models determination of the presence of metastatic disease, metastatic burden, and response to therapy are difficult, often requiring serial necropsy. In this study we characterized the development of visceral metastases in a patient derived xenograft model (PDXM) using in vivo imaging. RESULTS: We identified and characterized the previously unreported development of spontaneous liver and bone metastasis in a known patient derived xenograft, bladder xenograft BL0293F, developed by Jackson Laboratories and the University of California at Davis and available from the National Cancer Institute Patient-Derived Models Repository [1]. Among FDG-PET/CT, contrast-enhanced MRI and non-contrast MRI, non-contrast T2w MRI was the most effective and efficient imaging technique. On non-contrast T2 weighted MRI, hepatic metastases were observed in over 70% of animals at 52 days post tumor implantation without resection of the xenograft and in 100% of animals at day 52 following resection of the xenograft. In a group of animals receiving one cycle of effective chemotherapy, no animals demonstrated metastasis by imaging, confirming the utility of this model for therapy evaluation. There was good agreement between pathologic grade and extent of involvement observed on MRI T2w imaging. CONCLUSION: PDX BL0293F is a reliable visceral organ (liver) metastatic model with high penetrance in both non-aggravated and post excisional situations, providing a reliable window for therapy intervention prior to required excision of the xenograft. The imaging characteristics of this model are highly favorable for non-clinical research studies of metastatic disease when used in conjunction with non-contrast T2 weighted MRI.

Subcutaneous Alfaxalone-XylazineBuprenorphine for Surgical Anesthesia and Echocardiographic Evaluation of Mice (Mus musculus).

Alfaxalone is a commonly used injectable anesthetic in dogs and cats due to its minimal cardiovascular side effects. Data for its use in mice are limited and demonstrate strain- and sex-associated differences in dose-response relationships. We performed a dose-comparison study of alfaxalone-xylazine-buprenorphine (AXB) in Crl: CFW (SW) mice. Subcutaneous injection of 50 mg/kg alfaxalone-10 mg/kg xylazine-0.1 mg/kg buprenorphine HCl consistently achieved a surgical plane of anesthesia (loss of toe pinch) for 48.6 ± 4.7 and 60.8 ± 9.6 min in females and males, respectively. The same dose and route of AXB induced a surgical plane of anesthesia in C57Bl/6NCrl (females: 42.3 ± 11.2 min; males: 51.6 ± 12.3 min), NCr-Foxn1nu (females: 76.8 ± 32.5 min; males: 80.0 ± 1.2 min), and NOD. Cg-Prkdc SCID Il2rg tm1Wjl /SzJCr (females: 56.0 ± 37.2 min and males: 61.2 ± 10.2 min) mice. We found no significant difference in the duration of the surgical plane of anesthesia between males and females within the mouse strains Crl: CFW (SW), C57Bl/6NCrl, NCr-Foxn1nu, and NOD. Cg-PrkdcSCID Il2rgtm1Wjl /SzJCr. We next performed an echocardiography study (n = 5 per group) of Crl: CFW (SW) mice ( n = 5 per group) to compare subcutaneous AXB anesthesia with that produced by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine (KX). AXB induced significantly less bradycardia (295.4 ± 29 bpm) than KX (185.8 ± 38.9 bpm) did, with no significant differences in cardiac output, ejection fraction, end-diastolic volume, end-systolic volume, or fractional shortening. These results suggest that subcutaneous administration of AXB is a viable alternative to KX for inducing a surgical plane of anesthesia in Crl: CFW (SW), C57Bl/6NCrl, NCr-Foxn1nu, and NOD. Cg-PrkdcSCID Il2rgtm1Wjl /SzJCr mice, regardless of sex. AXB may also be a better injectable anesthetic option as compared with KX for avoiding adverse cardiac effects in mice.

A dual-luciferase bioluminescence system for the assessment of cellular therapies.

Bioluminescence imaging is a well-established platform for evaluating engineered cell therapies in preclinical studies. However, despite the discovery of new luciferases and substrates, optimal combinations to simultaneously monitor two cell populations remain limited. This makes the functional assessment of cellular therapies cumbersome and expensive, especially in preclinical in vivo models. In this study, we explored the potential of using a green bioluminescence-emitting click beetle luciferase, CBG99, and a red bioluminescence-emitting firefly luciferase mutant, Akaluc, together to simultaneously monitor two cell populations. Using various chimeric antigen receptor T cells and tumor pairings, we demonstrate that these luciferases are suitable for real-time tracking of two cell types using 2D and 3D cultures in vitro and experimental models in vivo. Our data show the broad compatibility of this dual-luciferase (duo-luc) system with multiple bioluminescence detection equipment ranging from benchtop spectrophotometers to live animal imaging systems. Although this study focused on investigating complex CAR T cells and tumor cell interactions, this duo-luc system has potential utility for the simultaneous monitoring of any two cellular components-for example, to unravel the impact of a specific genetic variant on clonal dominance in a mixed population of tumor cells.

Preclinical Therapeutic Efficacy of RAF/MEK/ERK and IGF1R/AKT/mTOR Inhibition in Neuroblastoma.

Activating mutations in the RAS/MAPK pathway are observed in relapsed neuroblastoma. Preclinical studies indicate that these tumors have an increased sensitivity to inhibitors of the RAS/MAPK pathway, such as MEK inhibitors. MEK inhibitors do not induce durable responses as single agents, indicating a need to identify synergistic combinations of targeted agents to provide therapeutic benefit. We previously showed preclinical therapeutic synergy between a MEK inhibitor, trametinib, and a monoclonal antibody specific for IGF1R, ganitumab in RAS-mutated rhabdomyosarcoma. Neuroblastoma cells, like rhabdomyosarcoma cells, are sensitive to the inhibition of the RAS/MAPK and IGF1R/AKT/mTOR pathways. We hypothesized that the combination of trametinib and ganitumab would be effective in RAS-mutated neuroblastoma. In this study, trametinib and ganitumab synergistically suppressed neuroblastoma cell proliferation and induced apoptosis in cell culture. We also observed a delay in tumor initiation and prolongation of survival in heterotopic and orthotopic xenograft models treated with trametinib and ganitumab. However, the growth of both primary and metastatic tumors was observed in animals receiving the combination of trametinib and ganitumab. Therefore, more preclinical work is necessary before testing this combination in patients with relapsed or refractory RAS-mutated neuroblastoma.

Targeting NAD+ Metabolism Vulnerability in FH-Deficient Hereditary Leiomyomatosis and Renal Cell Carcinoma with the novel NAMPT Inhibitor OT-82.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome caused by germline pathogenic variants in the fumarate hydratase (FH) gene. Affected individuals are at risk for developing cutaneous and uterine leiomyomas and aggressive FH-deficient renal cell carcinoma (RCC) with a papillary histology. Due to a disrupted TCA cycle, FH-deficient kidney cancers rely on aerobic glycolysis for energy production, potentially creating compensatory metabolic vulnerabilities. This study conducted a high-throughput drug screen in HLRCC cell lines, which identified a critical dependency on nicotinamide adenine dinucleotide (NAD), a redox cofactor produced by the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). Human HLRCC tumors and HLRCC-derived cell lines exhibited elevated NAMPT expression compared to controls. FH-deficient HLRCC cells, but not FH-restored HLRCC or normal kidney cells, were sensitive to NAMPT inhibition. HLRCC cell line viability was significantly decreased in both 2D and 3D in vitro cultures in response to the clinically relevant NAMPT inhibitor OT-82. NAMPT inhibition in vitro significantly decreased the total amount of NAD+, NADH, NADP, NADPH, and PAR levels and the effects of NAMPT inhibition could be rescued by the downstream NAD precursor nicotinamide mononucleotide, confirming the on-target activity of OT-82. Moreover, NAMPT inhibition by OT-82 in two HLRCC xenograft models resulted in severely reduced tumor growth. OT-82 treatment of HLRCC xenograft tumors in vivo inhibited glycolytic flux as demonstrated by reduced lactate/pyruvate ratio in hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging experiments. Overall, our data define NAMPT inhibition as a potential therapeutic approach for FH-deficient HLRCC-associated renal cell carcinoma.

Metastatic organotropism in small cell lung cancer.

Metastasis is the leading cause of cancer-related deaths, yet its regulatory mechanisms are not fully understood. Small-cell lung cancer (SCLC) is the most metastatic form of lung cancer, with most patients presenting with widespread disease, making it an ideal model for studying metastasis. However, the lack of suitable preclinical models has limited such studies. We utilized well-annotated rapid autopsy-derived tumors to develop xenograft models that mimic key features of SCLC, including histopathology, rapid and widespread development of metastasis to the liver, brain, adrenal, bone marrow, and kidneys within weeks, and response to chemotherapy. By integrating in vivo lineage selection with comprehensive transcriptomic and epigenomic analyses, we identified critical cellular programs driving metastatic organotropism to the liver and brain, the most common sites of SCLC metastasis. Our findings reveal the key role of nuclear-cytoskeletal interactions in SCLC liver metastasis. Specifically, the loss of the nuclear envelope protein lamin A/C, encoded by the LMNA gene, increased nuclear deformability and significantly increased the incidence of liver metastasis. Human liver metastases exhibited reduced LMNA expression compared to other metastatic sites, correlating with poorer patient outcomes and increased mortality. This study introduces novel preclinical models for SCLC metastasis and highlights pathways critical for organ-specific metastasis, offering new avenues for the development of targeted therapies to prevent or treat metastatic disease.

Guidance for methods descriptions used in preclinical imaging papers.

Preclinical molecular imaging is a rapidly growing field, where new imaging systems, methods, and biological findings are constantly being developed or discovered. Imaging systems and the associated software usually have multiple options for generating data, which is often overlooked but is essential when reporting the methods used to create and analyze data. Similarly, the ways in which animals are housed, handled, and treated to create physiologically based data must be well described in order that the findings be relevant, useful, and reproducible. There are frequently new developments for metabolic imaging methods. Thus, specific reporting requirements are difficult to establish; however, it remains essential to adequately report how the data have been collected, processed, and analyzed. To assist with future manuscript submissions, this article aims to provide guidelines of what details to report for several of the most common imaging modalities. Examples are provided in an attempt to give comprehensive, succinct descriptions of the essential items to report about the experimental process.

Cross-neutralizing protection of vaginal and oral mucosa from HPV challenge by vaccination in a mouse model.

The species and tissue specificities of HPV (human papillomavirus) for human infection and disease complicates the process of prophylactic vaccine development in animal models. HPV pseudoviruses (PsV) that carry only a reporter plasmid have been utilized in vivo to demonstrate cell internalization in mouse mucosal epithelium. The current study sought to expand the application of this HPV PsV challenge model with both oral and vaginal inoculation and to demonstrate its utility for testing vaccine-mediated dual-site immune protection against several HPV PsV types. We observed that passive transfer of sera from mice vaccinated with the novel experimental HPV prophylactic vaccine RG1-VLPs (virus-like particles) conferred HPV16-neutralizing as well as cross-neutralizing Abs against HPV39 in naïve recipient mice. Moreover, active vaccination with RG1-VLPs also conferred protection to challenge with either HPV16 or HPV39 PsVs at both vaginal and oral sites of mucosal inoculation. These data support the use of the HPV PsV challenge model as suitable for testing against diverse HPV types at two sites of challenge (vaginal vault and oral cavity) associated with the origin of the most common HPV-associated cancers, cervical cancer and oropharyngeal cancer.

Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

Modified norcyanines enable ratiometric pH imaging beyond 1000 nm.

Activatable fluorophores with emission beyond 1000 nm have the potential to enable high contrast imaging in complex in vivo settings. However, there are few scaffolds that can be applied to this challenge. Here we detail the synthesis and evaluation of benzo[c,d]indole-substituted norcyanines that enable pH responsive fluorescence imaging in the long wavelength (>1150 nm) range. A key component of our molecular design is the installation of a hydrophilic substituted quaternary amine in the central dihydropyridine ring system. A compound with a C4'-phenyl substituent, but not the C4'-protio homologue, exhibits absorbance maxima of 740 nm and 1130 nm in basic and acidic media, respectively, with evidence of J-aggregate-like properties. These two distinct absorbances enabled ratiometric imaging of probe internalization in a tumor model. Overall, these studies provide a new class of activatable long-wavelength responsive fluorophores with promising photophysical properties.

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration.

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

Design and Implementation of the Pre-Clinical DICOM Standard in Multi-Cohort Murine Studies.

The small animal imaging Digital Imaging and Communications in Medicine (DICOM) acquisition context structured report (SR) was developed to incorporate pre-clinical data in an established DICOM format for rapid queries and comparison of clinical and non-clinical datasets. Established terminologies (i.e., anesthesia, mouse model nomenclature, veterinary definitions, NCI Metathesaurus) were utilized to assist in defining terms implemented in pre-clinical imaging and new codes were added to integrate the specific small animal procedures and handling processes, such as housing, biosafety level, and pre-imaging rodent preparation. In addition to the standard DICOM fields, the small animal SR includes fields specific to small animal imaging such as tumor graft (i.e., melanoma), tissue of origin, mouse strain, and exogenous material, including the date and site of injection. Additionally, the mapping and harmonization developed by the Mouse-Human Anatomy Project were implemented to assist co-clinical research by providing cross-reference human-to-mouse anatomies. Furthermore, since small animal imaging performs multi-mouse imaging for high throughput, and queries for co-clinical research requires a one-to-one relation, an imaging splitting routine was developed, new Unique Identifiers (UID's) were created, and the original patient name and ID were saved for reference to the original dataset. We report the implementation of the small animal SR using MRI datasets (as an example) of patient-derived xenograft mouse models and uploaded to The Cancer Imaging Archive (TCIA) for public dissemination, and also implemented this on PET/CT datasets. The small animal SR enhancement provides researchers the ability to query any DICOM modality pre-clinical and clinical datasets using standard vocabularies and enhances co-clinical studies.

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.