Accordion waves in Myxococcus xanthus

Myxococcus xanthus are Gram-negative bacteria that glide on solid surfaces, periodically reversing their direction of movement. When starved, M. xanthus cells organize their movements into waves of cell density that sweep over the colony surface. These waves are unique: Although they appear to interpenetrate, they actually reflect off one another when they collide, so that each wave crest oscillates back and forth with no net displacement. Because the waves reflect the coordinated back and forth oscillations of the individual bacteria, we call them “accordion” waves. The spatial oscillations of individuals are a manifestation of an internal biochemical oscillator, probably involving the Frz chemosensory system. These internal “clocks,” each of which is quite variable, are synchronized by collisions between individual cells using a contact-mediated signal-transduction system. The result of collision signaling is that the collective spatial behavior is much less variable than the individual oscillators. In this work, we present experimental observations in which individual cells marked with GFP can be followed in groups of unlabeled cells in monolayer cultures. These data, together with an agent-based computational model demonstrate that the only properties required to explain the ripple patterns are an asymmetric biochemical limit cycle that controls direction reversals and asymmetric contact-induced signaling between cells: Head-to-head signaling is stronger than head-to-tail signaling. Together, the experimental and computational data provide new insights into how populations of interacting oscillators can synchronize and organize spatially to produce morphogenetic patterns that may have parallels in higher organisms

XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.

The limitations of cancer cell lines have led to the development of direct patient-derived xenograft models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg(-/-) (NSG) mouse on which a patients tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice-an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice and provide a more accurate tumor model to guide patient treatment

Resistance to ROS1 Inhibition Mediated by EGFR Pathway Activation in Non-Small Cell Lung Cancer

<div><p>The targeting of oncogenic ‘driver’ kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.</p></div

EGF stimulation desensitizes parental HCC78 cells and CUTO-2 cells to ROS1 inhibition.

<p>(A) Parental HCC78 and HCC78-TR cells were treated with TAE684 for 3 days with or without the addition of 100<b> </b>ng/mL EGF and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3). Calculated IC₅₀ values for TAE684: parental+vehicle = 0.18 µM, parental+EGF = 0.57 µM, HCC78-TR+vehicle = 1.39 µM, and HCC78-TR+EGF = 1.45 µM. EGF significantly desensitized parental HCC78 but not HCC78-TR cells to TAE684 as determined by student’s paired t-test (p<0.05). (B) Parental HCC78 cells were treated with TAE684 for 4 hours, EGF for 10 minutes, or a combination of both. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. (C) CUTO-2 cells were treated with TAE684 for 4 days with or without the addition of 100<b> </b>ng/mL EGF and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3). Calculated IC₅₀ values for TAE684: +vehicle = 0.2 µM and +EGF = 0.81 µM. EGF significantly desensitized CUTO-2 cells to TAE684 as determined by student’s paired t-test (p<0.01). (D) CUTO-2 cells were treated with TAE684 for 4 hours, EGF for 10 minutes, or a combination of both. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. Phosphorylated ROS1 bands were below the limit of detection and were therefore not included.</p

HCC78-TR cells are resistant to ROS1 inhibition.

<p>Cells were treated with TAE684 (A), crizotinib (B), or pemetrexed (C) as single-agents for 3 days and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3–7). Calculated IC₅₀ values for TAE684: parental HCC78 = 0.14 µM, HCC78-TR = 1.09 µM, H322 = 1.42 µM, and HCC4006 = 1.15 µM. Calculated IC₅₀ values for crizotinib: parental HCC78 = 0.79 µM, HCC78-TR = 1.95 µM, H322 = 4.13 µM, and HCC4006 = 3.03 µM. Calculated IC₅₀ values for pemetrexed: parental HCC78 = 11<b> </b>nM and HCC78-TR = 14<b> </b>nM. HCC78-TR cells were significantly less sensitive than parental HCC78 cells to TAE684 (p<0.000005) and crizotinib (p<0.05) but not pemetrexed (p>0.05) as determined by student’s paired t-test.</p

HCC78-TR cells have become sensitive to EGFR inhibition.

<p>(A) Parental HCC78 and HCC78-TR cells were treated with gefitinib as a single-agent for 3 days and then analyzed by MTS assay. (B) Parental HCC78 and HCC78-TR cells were co-treated with 500<b> </b>nM TAE684 and gefitinib, and HCC827, H322, and H358 cells were treated with single-agent gefitinib for 3 days and then analyzed by MTS assay. Calculated IC₅₀ values: parental HCC78 (below 50% with single-agent TAE684), HCC78-TR = 0.86 µM, HCC827 = 0.04 µM, H322 = >5 µM, and H358 = 1.0<b> </b>uM. All values represent the mean ± SEM (n = 4).</p