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The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging, given the complex tumor microenvironment including intra- and intertumor heterogeneity. The difficulty in studying this distribution is even more significant for small-molecule drugs where radiolabeled compounds or mass spectrometry detection lack the spatial and temporal resolution required to quantify the kinetics of drug distribution in vivo. In this work, we take advantage of the synthesis of fluorescent drug conjugates that retain their target binding but are designed with different physiochemical and thus pharmacokinetic properties. Using these probes, we followed the drug distribution in cell culture and tumor xenografts with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small-molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents as demonstrated by a finite element model. Mol Cancer Ther; 13(4); 986–95. ©2014 AACR.

PDF file - 401KB, Supplemental file containing a mathematical model description, Figure S1 showing the modeling approach, Table S1 with model parameters, and Figure S2 graphing cellular clearance.

Acute radiation syndrome (ARS) is the radiation toxicity that can affect the hematopoietic, gastrointestinal, and nervous systems upon accidental radiation exposure within a short time. Currently, there are no effective and safe approaches to treat mass population exposure to ARS. Our study aimed to evaluate the therapeutic potential of allogeneic adipose-derived stem cells (ASCs) for total body irradiation (TBI)-induced ARS and understand the underlying mitigation mechanism. We employed 9.25 Gy TBI dose to C57BL/6 mice and studied the effect of allogeneic ASCs on mice survival and regeneration of the hematopoietic system. Our results indicate that intraperitoneal-injected ASCs migrated to the bone marrow, rescued hematopoiesis, and improved the survival of irradiated mice. Our transwell coculture results confirmed the migration of ASCs to irradiated bone marrow and rescue hematopoietic activity. Furthermore, contact coculture of ASCs improved the survival and hematopoiesis of irradiated bone marrow in vitro. Irradiation results in DNA damage, upregulation of inflammatory signals, and apoptosis in bone marrow cells, while coculture with ASCs reduces apoptosis via activation of DNA repair and the antioxidation system. Upon exposure to irradiated bone marrow cells, ASCs secrete prosurvival and hematopoietic factors, such as GM-CSF, MIP1α, MIP1β, LIX, KC, 1P-10, Rantes, IL-17, MCSF, TNFα, Eotaxin, and IP-10, which reduces oxidative stress and rescues damaged bone marrow cells from apoptosis. Our findings suggest that allogeneic ASCs therapy is effective in mitigating TBI-induced ARS in mice and may be beneficial for clinical adaptation to treat TBI-induced toxicities. Further studies will help to advocate the scale-up and adaptation of allogeneic ASCs as the radiation countermeasure.

Tumor cell derived extracellular vesicles (EV) are being explored as circulating biomarkers for cancer detection. Up to now however, clinical results have been mixed for a number of reasons including the predominant use of bulk measurements, the inability to differentiate tumor from host cell derived vesicles, the general absence of uniquely identifying biomarkers and the unknown frequency of stochastically distributed biomarkers into single circulating vesicles. We hypothesized that a single EV analysis (sEVA) technique could potentially improve diagnostic accuracy necessary to detect early cancers but the actual biomarker frequency and practical detection limits are currently unknown. Using pancreatic cancer, we carefully analyzed the composition of putative cancer markers in 11 established and new patient derived models. In parental PDAC cells positive for KRASmutand/or P53mutproteins only ∼40% of EVs were also positive (range: 30-64%). This rate of positivity increased to 57% when additional PDAC biomarkers were considered (MUC1, EGFR, ⍺FG-P4OH) in cell lines. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRASmutand P53mutprotein was detectable at much lower levels, generally in < 0.1% of vesicles. With the analytical capabilities of sEVA however, 15 of the 16 patients with stage 1 PDAC expressed low levels of biomarker positive EV. Using a modeling approach, we estimate that the current PDAC detection limit is at ∼0.1 cm3tumor volume, below clinical imaging capabilities. These findings establish the potential for single-EV analysis for early cancer detection.

The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb. Replacement of V(D)J recombinase targets at two different mouse Vβ gene segments with a higher quality target elevates Vβ rearrangement frequency before feedback inhibition, dramatically increasing the frequency of T cells with TCRβ chains derived from both Tcrb alleles. Thus, TCRβ allelic exclusion is enforced genetically by the low quality of Vβ recombinase targets that stochastically restrict the production of two functional rearrangements before feedback inhibition silences one allele.

SUMMARYMonoallelic expression (allelic exclusion) of T and B lymphocyte antigen receptor genes is achieved by the assembly of a functional gene through V(D)J recombination on one allele and subsequent feedback inhibition of recombination on the other allele. There has been no validated mechanism for how only one allele of any antigen receptor locus assembles a functional gene prior to feedback inhibition. Here, we demonstrate that replacement of a single Vβ recombination signal sequence (RSS) with a better RSS increases Vβ rearrangement, revealsTcrballeles compete for utilization in the αβ T cell receptor (TCR) repertoire, and elevates the fraction of αβ T cells expressing TCRβ protein from both alleles. The data indicate that poor qualities of Vβ RSSs for recombination with Dβ and Jβ RSSs enforces allelic exclusion by stochastically limiting the incidence of functional Vβ rearrangements on both alleles before feedback inhibition terminates Vβ recombination.

Adipose tissue plays an important role in regulating metabolic homeostasis by storing excess fat and protecting other organs from lipotoxicity. Aging is associated with central fat redistribution, culminating in a decrease in insulin-sensitive subcutaneous and an increase in insulin-resistant visceral adipose depots. Adipose-derived stem cells (ASCs) play an important role in the regeneration of adipose tissue. Aged ASCs show decreased stemness and regenerative potential due to the accumulation of oxidative stress and mitochondrial dysfunction-related cell damage. Metformin is a well-established anti-diabetic drug that has shown anti-aging effects in different organisms and animal models. In this study, we analyzed the effect of metformin treatment on the stemness of human ASCs in cell culture and whole adipose tissue culture models. Our results demonstrate that metformin improves the stemness of ASCs, reducing their rate of proliferation and adipocyte differentiation. Investigating the possible underlying mechanism, we observed a decrease in the mTOR and ERK activity in metformin-treated ASCs. In addition, we observed an increase in autophagy activity upon metformin treatment. We conclude that metformin treatment improves ASCs stemness by reducing mTOR and ERK signaling and enhancing autophagy. Future in vivo evaluations in animal models and humans will pave the way for the clinical adaptation of this well-established drug for reviving the stemness of aged stem cells.

Background and Aims Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs), and their management remains controversial. At present, there is no reliable noninvasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions. Methods Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST), we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n = 86) and a validation cohort (n = 47). Results From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high-grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV of the 11 patients with high-grade dysplasia alone, only 1 had high MUC5AC expression (sensitivity of 82%, specificity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high-risk stigmata allowed detection of all cases requiring surgery, whereas imaging and high-risk stigmata alone would have missed 5 of 14 cases (36%). Conclusions MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATMflox) was crossed to hypomorphic NBS1 mutants (Nbs1ΔB/ΔB mice). Nbs1ΔB/ΔB Atm−/− hematopoietic cells derived by crossing to vavcre were viable in vivo. Nbs1ΔB/ΔB Atm−/− VAV mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. Implications: A novel mouse model was developed, by combining a vavcre-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. Mol Cancer Res; 14(2); 185–95. ©2015 AACR.

Background: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. Methods: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell&ndash, cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. Results: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell&ndash, cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. Conclusion: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.

Extracellular vesicles (EVs) represent promising circulating biomarkers for cancers, but their high-throughput analyses in clinical settings prove challenging due to lack of simple, fast, and robust EV assays. Here, we describe a bead-based EV assay detected by flow cytometry, which integrates EV capture using microbeads with EV protein analyses by flow cytometry. The assay is fast (< 4 hrs for 48 samples), robust and compatible with conventional flow cytometry instruments for high-throughput EV analysis. With the method, we successfully analyzed a panel of pancreatic cancer biomarkers in EVs from plasma samples of pancreatic cancer patients. The assay is readily translatable to other biomarkers or cancer types and can be run with standard materials on conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

Bruton's tyrosine kinase (Btk) is intricately involved in anti-apoptotic signaling pathways in cancer and in regulating innate immune response. A number of Btk inhibitors are in development for use in treating B-cell malignancies and certain immunologic diseases. To develop robust companion imaging diagnostics for in vivo use, we set out to explore the effects of red wavelength fluorochrome modifications of two highly potent irreversible Btk inhibitors, Ibrutinib and AVL-292. Surprisingly, we found that subtle chemical differences in the fluorochrome had considerable effects on target localization. Based on iterative designs, we developed a single optimized version with superb in vivo imaging characteristics enabling single cell Btk imaging in vivo. This agent (Ibrutinib-SiR-COOH) is expected to be a valuable chemical tool in deciphering Btk biology in cancer and host cells in vivo.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of immature T cells that exhibits heterogeneity of oncogenic lesions, providing an obstacle for development of more effective and less toxic therapies. Inherited deficiency of ATM, a regulator of the cellular DNA damage response, predisposes young humans and mice to T-ALLs with clonal chromosome translocations. While acquired ATM mutation or deletion occurs in pediatric T-ALLs, the role of somatic ATM alterations in T-ALL pathogenesis remains unknown. We demonstrate here that somatic Atm inactivation in haematopoietic cells starting as these cells differentiate in utero predisposes mice to T-ALL at similar young ages and harboring analogous translocations as germline Atm-deficient mice. However, some T-ALLs from haematopoietic cell specific deletion of Atm were of more mature thymocytes, revealing that the developmental timing and celluar origin of Atm inactivation influences the phenotype of ATM-deficient T-ALLs. Although it has been hypothesized that ATM suppresses cancer by preventing deletion and inactivation of TP53, we find that Atm inhibits T-ALL independent of Tp53 deletion. Finally, we demonstrate that the Cyclin D3 protein that drives immature T cell proliferation is essential for transformation of Atm-deficient thymocytes. Our study establishes a pre-clinical model for pediatric T-ALLs with acquired ATM inactivation and identifies the cell cycle machinery as a therapeutic target for this aggressive childhood T-ALL subtype.

Monoclonal antibodies (mAbs) targeting the immune checkpoint anti–programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1 + tumor-infiltrating CD8 + T cells at early time points after administration. However, this engagement is transient, and aPD-1 mAbs are captured within minutes from the T cell surface by PD-1 − tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug’s Fc domain glycan and on Fcγ receptors (FcγRs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs before aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8 + T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.

Majumder et al. explore the large-scale looping architecture of the Tcrb locus early in murine thymocyte development during the generation of TCRβ diversity. They dissect novel DNA regulatory elements controlling V to D-J recombination and identify within an insulator region a distally located CTCF-containing element functioning as a tether, which facilitates looping of distal Vβ to Dβ-Jβ regions and promotes locus contraction. A second CTCF-containing element, proximal to the Dβ-Jβ region, acts as a boundary, preventing the spread of active chromatin associated with Dβ-Jβ regions. Removal of the proximal boundary element impairs the locus contraction capabilities of the tethering element., Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb). Association between distal Vβ gene segments and the highly expressed DβJβ clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vβ array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers.

Overexpression of anti-apoptotic proteins such as Bcl-2 is a cellular mechanism to evade apoptosis; consequently, Bcl-2 inhibitors are being developed as anticancer agents. In this work, we have synthesized a fluorescent version of ABT-199 in an effort to visualize a drug surrogate by high resolution imaging. We show that this fluorescent conjugate has comparable Bcl-2 binding efficacy and cell line potency to the parent compound and can be used as an imaging agent in several cancer cell types. We anticipate that this agent will be a valuable tool for studying the single-cell distribution and pharmacokinetics of ABT-199 as well the broader group of BH3-mimetics.

ATM is the master regulator of the cellular response to DNA double strand breaks (DSBs). Deficiency of ATM predisposes humans and mice to αβ T lymphoid cancers with clonal translocations between the T cell receptor (TCR) α/δ locus and a 450 kb region of synteny on human chromosome 14 and mouse chromosome 12. While these translocations target and activate the TCL1 oncogene at 14q32 to cause T cell pro-lymphocytic leukemia (T-PLL), the TCRα/δ;14q32 translocations in ATM-deficient T cell acute lymphoblastic leukemia (T-ALL) have not been characterized and their role in cancer pathogenesis remains unknown. The corresponding lesion in Atm-deficient mouse T-ALLs is a chromosome t(12;14) translocation with Tcrδ genes fused to sequences on chromosome 12; although these translocations do not activate Tcl1, they delete the Bcl11b haploinsufficient tumor suppressor gene. To assess whether Tcrδ translocations that inactivate one copy of Bcl11b promote transformation of Atm-deficient cells, we analyzed Atm(-/-) mice with mono-allelic Bcl11b deletion initiating in thymocytes concomitant with Tcrδ recombination. Inactivation of one Bcl11b copy had no effect on the predisposition of Atm(-/-) mice to clonal T-ALLs. Yet, none of these T-ALLs had a clonal chromosome t(12;14) translocation that deleted Bcl11b indicating that Tcrδ translocations that inactivate a copy of Bcl11b promote transformation of Atm-deficient thymocytes. Our data demonstrate that antigen receptor locus translocations can cause cancer by deleting a tumor suppressor gene. We discuss the implications of these findings for the etiology and therapy of T-ALLs associated with ATM deficiency and TCRα/δ translocations targeting the 14q32 cytogenetic region.

Platinum(II) compounds, principally cisplatin and carboplatin, are commonly used front-line cancer therapeutics. Despite their widespread use and continued interest in the development of new derivatives, including nanoformulations with improved properties, it has been difficult to visualize platinum compounds in live subjects, in real time, and with subcellular resolution. Here, we present four novel cisplatin- and carboplatin-derived fluorescent imaging compounds for quantitative intravital cancer imaging. We conjugated 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-daiza-s-indacene (BODIPY) to Pt(II) complexes to generate derivatives with robust in vivo fluorescence and retained DNA-damaging and cytotoxic properties. We successfully applied these compounds to image pharmacokinetics and tumor uptake in a xenograft cancer mouse model. By using a genetic reporter of single-cell DNA damage for in vivo imaging, Pt drug accumulation and resultant DNA damage could be monitored in individual tumor cells, at subcellular resolution, and in real time in a live animal model of cancer. These derivatives represent promising imaging tools that will be useful in understanding further the distribution and interactions of platinum within tumors.

Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel “pre-leukemic” stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers. Cancer Immunol Res; 1(6); 426–37. ©2013 AACR.

TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53(-/-) mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53(-/-) mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53(-/-) mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.

The DNA damage response (DDR) can restrain the ability of oncogenes to cause genomic instability and drive malignant transformation. The gene encoding the histone H2AX DDR factor maps to 11q23, a region frequently altered in human cancers. Since H2ax functions as a haploinsufficient suppressor of B lineage lymphomas with c-Myc amplification and/or translocation, we determined the impact of H2ax expression on the ability of deregulated c-Myc expression to cause genomic instability and drive transformation of B cells. Neither H2ax deficiency nor haploinsufficiency affected the rate of mortality of Eμ-c-Myc mice from B lineage lymphomas with genomic deletions and amplifications. Yet H2ax functioned in a dosage-dependent manner to prevent unbalanced translocations in Eμ-c-Myc tumors, demonstrating that H2ax functions in a haploinsufficient manner to suppress allelic imbalances and limit molecular heterogeneity within and among Eμ-c-Myc lymphomas. Regardless of H2ax copy number, all Eμ-c-Myc tumors contained identical amplification of chromosome 19 sequences spanning 20 genes. Many of these genes encode proteins with tumor-promoting activities, including Cd274, which encodes the PD-L1 programmed death ligand that induces T cell apoptosis and enables cancer cells to escape immune surveillance. This amplicon was in non-malignant B and T cells and non-lymphoid cells, linked to the Eμ-c-Myc transgene, and associated with overexpression of PD-L1 on non-malignant B cells. Our data demonstrate that, in addition to deregulated c-Myc expression, non-malignant B lineage lymphocytes of Eμ-c-Myc transgenic mice may have constitutive amplification and increased expression of other tumor-promoting genes.

A proteomics method to pull down secondary drug targets from live cells is described. The drug of interest is modified with trans-cyclooctene (TCO) and incubated with live cells. Upon cell lysis, the modified drug bound to the protein is pulled down using magnetic beads decorated with a cleavable tetrazine-modified linker. Samples are then run on an SDS-PAGE gel and isolated bands are submitted for mass spectrometry analysis to identify drug targets.

DNA double-strand breaks induced during Igκ recombination signal through ATM to suppress the initiation of additional Vκ-to-Jκ rearrangements., Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre–B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.

Pancreatic ductal adenocarcinoma (PDAC) is usually detected late in the disease process. Clinical workup through imaging and tissue biopsies is often complex and expensive due to a paucity of reliable biomarkers. We used an advanced multiplexed plasmonic assay to analyze circulating tumor-derived extracellular vesicles (tEVs) in more than 100 clinical populations. Using EV-based protein marker profiling, we identified a signature of five markers (PDACEV signature) for PDAC detection. In our prospective cohort, the accuracy for the PDACEV signature was 84% [95% confidence interval (CI), 69 to 93%] but only 63 to 72% for single-marker screening. One of the best markers, GPC1 alone, had a sensitivity of 82% (CI, 60 to 95%) and a specificity of 52% (CI, 30 to 74%), whereas the PDACEV signature showed a sensitivity of 86% (CI, 65 to 97%) and a specificity of 81% (CI, 58 to 95%). The PDACEV signature of tEVs offered higher sensitivity, specificity, and accuracy than the existing serum marker (CA 19-9) or single-tEV marker analyses. This approach should improve the diagnosis of pancreatic cancer.

Mitochondria, which are essential organelles in resting and replicating cells, can vary in number, mass and shape. Past research has primarily focused on short-term molecular mechanisms underlying fission/fusion. Less is known about longer-term mitochondrial behavior such as the overall makeup of cell populations’ morphological patterns and whether these patterns can be used as biomarkers of drug response in human cells. We developed an image-based analytical technique to phenotype mitochondrial morphology in different cancers, including cancer cell lines and patient-derived cancer cells. We demonstrate that (i) cancer cells of different origins, including patient-derived xenografts, express highly diverse mitochondrial phenotypes; (ii) a given phenotype is characteristic of a cell population and fairly constant over time; (iii) mitochondrial patterns correlate with cell metabolic measurements and (iv) therapeutic interventions can alter mitochondrial phenotypes in drug-sensitive cancers as measured in pre- versus post-treatment fine needle aspirates in mice. These observations shed light on the role of mitochondrial dynamics in the biology and drug response of cancer cells. On the basis of these findings, we propose that image-based mitochondrial phenotyping can provide biomarkers for assessing cancer phenotype and drug response.

Small molecule imaging: Aurora kinase A (AKA) was imaged in live cells using an in cellulo bioorthogonal two-step reaction with a small molecule and a fluorescent reporter. The small molecule was localized to spindle poles and microtubules during metaphase, consistent with the localization of both endogenous and GFP-/RFP-tagged AKA. Using this approach, changes in AKA distribution during mitosis were also observed.

Responses to molecularly targeted therapies can be highly variable and depend on mutations, fluctuations in target protein levels in individual cells, and drug delivery. The ability to rapidly quantitate drug response in cells harvested from patients in a point-of-care setting would have far reaching implications. Capitalizing on recent developments with miniaturized NMR technologies, we have developed a magnetic nanoparticle-based approach to directly measure both target expression and drug binding in scant human cells. The method involves covalent conjugation of the small-molecule drug to a magnetic nanoparticle that is then used as a read-out for target expression and drug-binding affinity. Using poly(ADP-ribose) polymerase (PARP) inhibition as a model system, we developed an approach to distinguish differential expression of PARP in scant cells with excellent correlation to gold standards, the ability to mimic drug pharmacodynamics ex vivo through competitive target-drug binding, and the potential to perform such measurements in clinical samples.

A nuclear protein target, polo-like kinase 1 (PLK1) was imaged using a biocompatible bioorthogonal ligation between a specific drug and a fluorescent dye in live cells. Colocalization of the dye and the protein target was confirmed by antibody staining and by expressing a GFP construct of PLK1. The two-step PLK1 imaging procedure was used to quantify PLK1 expression levels in cancer cell lines of various tissue origins.

The epidermal growth factor (EGF) receptor is a receptor tyrosine kinase involved in the control of cell proliferation, and its overexpression is strongly associated with a variety of aggressive cancers. For example, 70-80% of metaplastic (cancer cells of mixed type) breast carcinomas overexpress EGF receptors. In addition, the EGF receptor is a highly significant contributor to common brain tumors (glioblastoma multiforme), both in initiation and progression (Huang P.H., Xu A.M., White F.M. (2009) Oncogenic EGFR signaling networks in glioma. Sci Signal;2:re6.). Brain metastases, an unmet medical need, are also common in metastatic cancer associated with overexpression of EGF receptors. Formation of EGF receptor homodimers is essential for kinase activation and was the basis for exploring direct inhibition of EGF receptor activation by blocking dimerization with small molecules. While inhibitors of protein/protein interactions are often considered difficult therapeutic targets, NSC56452, initially identified by virtual screening, was shown experimentally to inhibit EGF receptor kinase activation in a dose-dependent manner. This compound blocked EGF-stimulated dimer formation as measured by chemical cross-linking and luciferase fragment complementation. The compound was further shown to inhibit the growth of HeLa cells. This first-generation lead compound represents the first drug-like, small-molecule inhibitor of EGF receptor activation that is not directed against the intracellular kinase domain.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.

Crystal structures of the epidermal growth factor (EGF) receptor suggest that its activation is associated with extensive conformational changes in both the extracellular and intracellular domains. However, evidence of these structural dynamics in intact cells has been lacking. Here we use luciferase complementation imaging to follow EGF-induced conformational changes in its receptor in real time in live cells. When the luciferase fragments are fused to the C terminus of an EGF receptor lacking the cytoplasmic domain, EGF stimulates a rapid increase in luciferase activity, consistent with ligand-induced receptor dimerization. However, when the luciferase fragments are fused to the C terminus of the full-length receptor, EGF induces a rapid but transient decrease in luciferase activity. The decrease requires tyrosine kinase activity, whereas the subsequent recovery requires MAP kinase activity. Our data demonstrate the utility of the luciferase system for in vivo imaging changes in EGF receptor dimerization and conformation. They also identify two sequential ligand-induced conformational changes in the EGF receptor.

Therapeutic nanoparticles (TNPs) aim to deliver drugs more safely and effectively to cancers, yet clinical results have been unpredictable owing to limited in vivo understanding. Here we use single-cell imaging of intratumoral TNP pharmacokinetics and pharmacodynamics to better comprehend their heterogeneous behaviour. Model TNPs comprising a fluorescent platinum(IV) pro-drug and a clinically tested polymer platform (PLGA-b-PEG) promote long drug circulation and alter accumulation by directing cellular uptake toward tumour-associated macrophages (TAMs). Simultaneous imaging of TNP vehicle, its drug payload and single-cell DNA damage response reveals that TAMs serve as a local drug depot that accumulates significant vehicle from which DNA-damaging Pt payload gradually releases to neighbouring tumour cells. Correspondingly, TAM depletion reduces intratumoral TNP accumulation and efficacy. Thus, nanotherapeutics co-opt TAMs for drug delivery, which has implications for TNP design and for selecting patients into trials., Drug-loaded nanoparticles allow controlled release and enhanced delivery, yet understanding in vivo behavior has been difficult. Here, the authors develop a platinum prodrug coupled to a polymer platform, and use intravital imaging to show that the nanoparticle accumulates in macrophages, from the which drug redistributes to neighboring tumour cells.

Targeting DNA repair pathways is a powerful strategy to treat cancers. To gauge efficacy in vivo, typical response markers include late stage effects such as tumor shrinkage, progression free survival, or invasive repeat biopsies. These approaches are often difficult to answer critical questions such as how a given drug affects single cell populations as a function of dose and time, distance from microvessels or how drug concentration (pharmacokinetics) correlates with DNA damage (pharmacodynamics). Here, we established a single-cell in vivo pharmacodynamic imaging read-out based on a truncated 53BP1 double-strand break reporter to determine whether or not poly(ADP-ribose) polymerase (PARP) inhibitor treatment leads to accumulation of DNA damage. Using this reporter, we show that not all PARP inhibitor treated tumors incur an increase in DNA damage. The method provides a framework for single cell analysis of cancer therapeutics in vivo.

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vβ gene segments (Trbvs) and their DβJβ targets. Formation of a functional VβDβJβ join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DβJβ segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DβJβ segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VβDβJβ join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DβJβ targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation.

Therapeutic nanoparticles (TNPs) have shown heterogeneous responses in human clinical trials, raising questions of whether imaging should be used to identify patients with a higher likelihood of NP accumulation and thus therapeutic response. Despite extensive debate about the enhanced permeability and retention (EPR) effect in tumors, it is increasingly clear that EPR is extremely variable; yet, little experimental data exist to predict the clinical utility of EPR and its influence on TNP efficacy. We hypothesized that a 30-nm magnetic NP (MNP) in clinical use could predict colocalization of TNPs by magnetic resonance imaging (MRI). To this end, we performed single-cell resolution imaging of fluorescently labeled MNPs and TNPs and studied their intratumoral distribution in mice. MNPs circulated in the tumor microvasculature and demonstrated sustained uptake into cells of the tumor microenvironment within minutes. MNPs could predictably demonstrate areas of colocalization for a model TNP, poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG), within the tumor microenvironment with >85% accuracy and circulating within the microvasculature with >95% accuracy, despite their markedly different sizes and compositions. Computational analysis of NP transport enabled predictive modeling of TNP distribution based on imaging data and identified key parameters governing intratumoral NP accumulation and macrophage uptake. Finally, MRI accurately predicted initial treatment response and drug accumulation in a preclinical efficacy study using a paclitaxel-encapsulated NP in tumor-bearing mice. These approaches yield valuable insight into the in vivo kinetics of NP distribution and suggest that clinically relevant imaging modalities and agents can be used to select patients with high EPR for treatment with TNPs.

Eribulin mesylate was developed as a potent microtubule-targeting cytotoxic agent to treat taxane-resistant cancers, but recent clinical trials have shown that it eventually fails in many patient sub-populations for unclear reasons. To investigate its resistance mechanisms, we developed a fluorescent analog of eribulin with pharmacokinetic (PK) properties and cytotoxic activity across a human cell line panel that are sufficiently similar to the parent drug to study its cellular PK and tissue distribution. Using intravital imaging and automated tracking of cellular dynamics, we found that resistance to eribulin and the fluorescent analog depended directly on the multidrug resistance protein 1 (MDR1). Intravital imaging allowed for real-time analysis of in vivo pharmacokinetics in tumors that were engineered to be spatially heterogeneous for taxane resistance, whereby an MDR1-mApple fusion protein distinguished resistant cells fluorescently. In vivo, MDR1-mediated drug efflux and the three-dimensional tumor vascular architecture were discovered to be critical determinants of drug accumulation in tumor cells. We furthermore show that standard intravenous administration of a third-generation MDR1 inhibitor, HM30181, failed to rescue drug accumulation; however, the same MDR1 inhibitor encapsulated within a nanoparticle delivery system reversed the multidrug-resistant phenotype and potentiated the eribulin effect in vitro and in vivo in mice. Our work demonstrates that in vivo assessment of cellular PK of an anticancer drug is a powerful strategy for elucidating mechanisms of drug resistance in heterogeneous tumors and evaluating strategies to overcome this resistance.

The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging, given the complex tumor microenvironment including intra- and intertumor heterogeneity. The difficulty in studying this distribution is even more significant for small-molecule drugs where radiolabeled compounds or mass spectrometry detection lack the spatial and temporal resolution required to quantify the kinetics of drug distribution in vivo. In this work, we take advantage of the synthesis of fluorescent drug conjugates that retain their target binding but are designed with different physiochemical and thus pharmacokinetic properties. Using these probes, we followed the drug distribution in cell culture and tumor xenografts with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small-molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents as demonstrated by a finite element model. Mol Cancer Ther; 13(4); 986–95. ©2014 AACR.