Abstract. We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37°C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 I.tM.Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 ~tM -t s -t for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s -t. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 lxM. The rate of rapid shortening was similar at the two ends (plus = 733 s-t; minus = 915 s-l), and did not vary with tubulin concentration.Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.T HE term "dynamic instability" describes microtubule assembly in which individual microtubules exhibit alternating phases of elongation and rapid shortening. Transitions between these phases are abrupt, stochastic, and infrequent in comparison to the rates of tubulin association and dissociation at the microtubule ends (17, 28). Substantial evidence has accumulated to indicate that dynamic instability is the basic mechanism of microtubule assembly in vitro (17, 28), and in both the mitotic spindle and the cyoplasmic microtubule complex (CMTC) l (10,11,34,35,37). Although microtubule dynamics within the cell may be regulated by various microtubule-associated proteins (MAPs) and other intracellular regulatory molecules, it is important 1. Abbreviations used in this paper: CMTC, cytoplasmic microtubule complex; DIC, differential interference contrast; MAP(s), microtubule-associated protein(s).first to understand the details of the inherent behavior of microtubules assembled from tubulin alone.Th...

Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

Summary Blockade of colony-stimulating factor-1 (CSF-1) limits macrophage infiltration and improves response of mammary carcinomas to chemotherapy. Herein we identify interleukin (IL)-10 expression by macrophages as the critical mediator of this phenotype. Infiltrating macrophages were the primary source of IL-10 within tumors, and therapeutic blockade of IL-10 receptor (IL-10R) was equivalent to CSF-1 neutralization in enhancing primary tumor response to paclitaxel and carboplatin. Improved response to chemotherapy was CD8+ T cell-dependent, however IL-10 did not directly suppress CD8+ T cells or alter macrophage polarization. Instead, IL-10R blockade increased intratumoral dendritic cell expression of IL-12, which was necessary for improved outcomes. In human breast cancer, expression of IL12A and cytotoxic effector molecules were predictive of pathological complete response rates to paclitaxel.

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥ 50% of tumours expressing the BRAF(V600E) oncoprotein1,2. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance3,4. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy5. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xeno-graft models in which drugresistanceisselected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E) → MEK → ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8+ T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy.

No abstract

Abstract. Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 ~tm/min + 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 Ixm/min + 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s -~ for catastrophe and 0.044 s -~ for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 × 35 ~tm microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations. T HE great majority of cellular microtubules are labile polymers that exist in an equilibrium with a cytoplasmic pool of tubulin subunits (11,23,27). Dynamic microtubule polymers are essential for the rapid rearrangements of the microtubule cytoskeleton which occur during the transitions between the interphase and mitosis portions of the cell cycle. In addition, dynamic remodeling of microtubules is important in cell morphogenesis and directed cell motility (5,6,33).Although several models have been proposed to explain the mechanism of microtubule assembly behavior, studies of highly purified tubulin have identified dynamic instability as the mechanism of microtubule assembly dynamics in vitro (4,9,13,15,16,24,36). In this model microtubules exist in one of two phases: they are either elongating or rapidly shortening, with rare, abrupt, and random transitions between these phases. The entire microtubule population behaves heterogeneously because these transitions occur randomly; at any given time most microtubules will be growing at a relatively slow rate while fewer microtubules will be shortening at a relatively faster rate (15, 16).Recently, Horio and Hotani (9), Walker et al. light microscopic methods. These real-time observations have provided essential information on the dynamic instability behavior of microtubules that could not be obtained from analysis of samples fixed at time points. Detailed analysis of microtubule assembly by Walker et al. (36) has led to the following model of the dynamic history of a microtubule in vitro undergoing dynamic instability: a microtubule is initiated at a...

Abstract. Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 p,M) was assembled at 37"C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution) . Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length . Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated NDIVIDUAI. microtubules reassembled from purified tubulin undergo alternating phases ofelongation and rapid AL shortening (19,26,49) . The transition (catastrophe) from elongation to rapid shortening, and the reverse transition (rescue), are abrupt, stochastic, and occur infrequently in comparison to the rates of subunit association and dissociation (49) . This behavior, termed dynamic instability (26), is also exhibited by the majority ofmicrotubules in the mitotic spindle and the interphase cytoplasmic microtubule complex (CMTC) ' (12-14, 33-36, 38, 39) and appears necessary for chromosome-spindle attachment, chromosome segregation, cellular morphogenesis, and establishment ofcell polarity (13,17,21,(29)(30)(31)(32) . Changes in frequencies ofcatastrophe R . A . Walker's current address is