Single-Cell Computational Strategies for Lineage Reconstruction in Tissue SystemsSummary

Function at the organ level manifests itself from a heterogeneous collection of cell types. Cellular heterogeneity emerges from developmental processes by which multipotent progenitor cells make fate decisions and transition to specific cell types through intermediate cell states. Although genetic experimental strategies such as lineage tracing have provided insights into cell lineages, recent developments in single-cell technologies have greatly increased our ability to interrogate distinct cell types, as well as transitional cell states in tissue systems. From single-cell data that describe these intermediate cell states, computational tools have been developed to reconstruct cell-state transition trajectories that model cell developmental processes. These algorithms, although powerful, are still in their infancy, and attention must be paid to their strengths and weaknesses when they are used. Here, we review some of these tools, also referred to as pseudotemporal ordering algorithms, and their associated assumptions and caveats. We hope to provide a rational and generalizable workflow for single-cell trajectory analysis that is intuitive for experimental biologists. Keywords: Trajectory, Pseudotime, Single-Cell Analysis, Differentiation, Cell State Transition, Stem Cell

3'-Deoxy-3'-[18F]-Fluorothymidine PET imaging reflects PI3K-mTOR-mediated pro-survival response to targeted therapy in colorectal cancer.

Biomarkers that predict response to targeted therapy in oncology are an essential component of personalized medicine. In preclinical treatment response studies that featured models of wild-type KRAS or mutant BRAF colorectal cancer treated with either cetuximab or vemurafenib, respectively, we illustrate that [(18)F]-FLT PET, a non-invasive molecular imaging readout of thymidine salvage, closely reflects pro-survival responses to targeted therapy that are mediated by PI3K-mTOR activity. Activation of pro-survival mechanisms forms the basis of numerous modes of resistance. Therefore, we conclude that [(18)F]-FLT PET may serve a novel and potentially critical role to predict tumors that exhibit molecular features that tend to reflect recalcitrance to MAPK-targeted therapy. Though these studies focused on colorectal cancer, we envision that the results may be applicable to other solid tumors as well

Diminished TK1 protein levels correlate with attenuation of mTOR-PI3K pathway activity and upregulation of p27.

<p>(<b>A</b>) Western blot of DiFi cell lysates following cetuximab exposure (5 µg/mL) resulted in rapid attenuation of downstream MAPK targets including p-ERK, which remained well-below baseline levels to 24 hours. Paradoxically, TK1 protein levels increased from 1–12 hours until p27 protein levels rose, at which time TK1 fell bellow baseline. (<b>B</b>) DiFi cells treated with either 0.5 µg/mL or 5.0 µg/mL cetuximab resulted in a 50% reduction and full attenuation of p-ERK protein levels, respectively at 24 hours. At 0.5 µg/mL TK1 levels were unaffected despite a modest rise in p27 protein levels. However, 5.0 µg/mL cetuximab resulted in greatly decreased TK1 with a large increase in p27 protein levels. PI3K-mTOR activity, as measured by p-AKT Ser473, p-rpS6, and p-4E-BP1, was either maintained or elevated at 0.5 µg/mL cetuximab but was attenuated at 5.0 µg/mL cetuximab. (<b>C</b>) qRT-PCR analysis showed <i>TK1</i> mRNA was significantly reduced at 0.5 µg/mL (p = 0.0279) and 5.0 µg/mL (p = 0.0186), while no change in <i>p27</i> mRNA levels was observed at either dose. (<b>D</b>) Silencing mTORC1 facilitated upregulation of p27 and attenuated TK1 protein levels at 0.5 µg/mL cetuximab without affecting the anti-MAPK activity effects of cetuximab. Levels of p-AKT Ser473, p-rpS6, and p-4E-BP1 were reduced at 0.5 µg/mL cetuximab exposure with Raptor knockdown but not in scrambled siRNA control. (<b>E</b>) As evidence of the functionality of p27, <i>TK1</i> mRNA levels were similarly reduced at 0.5 µg/mL and 5.0 µg/mL with Raptor knockdown.</p

Combined ^V600EBRAF and mTOR inhibition results in transcriptional control of TK1 protein levels in COLO 205 cells.

<p>(<b>A</b>) Western blot of COLO 205 cells treated with PP242 (250 nM) and increasing PLX4032. Similar to single agent PLX4032, p-MEK, but not p-ERK, was inhibited in a PLX4032-dependent manner. Consistent with mTORC1/mTORC2 inhibition, p-AKT Ser473, but not p-AKT Thr308, was inhibited. Unlike single agent PLX4032, which resulted in concentration-dependent activation of p-rpS6, combined treatment maintained p-rpS6 levels at essentially baseline levels except at the highest PLX4032 concentration. Similarly, DUSP6 levels were inversely related to p-ERK protein levels. With combined mTOR and ^V600EBRAF blockade, p27 and TK1 protein levels were inversely correlated and dramatically affected by PLX4032 exposure. (<b>B</b>) Similarly, <i>TK1</i> mRNA was significantly reduced at PLX4032 concentrations as low as 10 nM. (<b>C</b>) Despite elevated p27 protein levels, <i>p27</i> mRNA was unaffected by combined mTOR-^V600EBRAF inhibition.</p

PLX4720 exposure does not affect [¹⁸F]-FLT PET in COLO 205 xenografts, despite evidence of target inhibition and diminished [¹⁸F]-FDG uptake.

<p>(<b>A</b>) Representative transverse [¹⁸F]-FLT and [¹⁸F]-FDG PET images acquired after three daily treatments with vehicle or 60 mg/kg PLX4720 (tumor indicated by arrowhead). (<b>B</b>) Quantification of PET data illustrated similar [¹⁸F]-FLT uptake in vehicle-treated and PLX4720-treated tumors. Unlike [¹⁸F]-FLT PET, PLX4720 exposure elicited a significant reduction in [¹⁸F]-FDG uptake (p = 0.0006). (<b>C</b>) Western blot analysis of vehicle- and PLX4720-treated tumor tissue confirmed that PLX4720 had no effect on TK1 protein levels in agreement with [¹⁸F]-FLT PET. Target inhibition as measured by p-MEK levels was observed. However, similar to <i>in vitro</i> studies, PLX4720-treated COLO 205 xenografts exhibited elevated p-ERK and p-rpS6 protein levels relative to vehicle controls.</p

Dual pathway inhibition regulates TK1 protein levels and results in greater p27 protein levels than single agents alone.

<p>Single agent PLX4032 resulted in activation of p-AKT Ser473 following 24 hours of exposure at two concentrations (100 nM, 1 µM). The addition of the dual PI3K/mTOR inhibitor BEZ235 blocks p-AKT Ser473 activation and resulted in a greater increase in p27 protein levels and diminished TK1 protein levels.</p

[¹⁸F]-FLT PET reflects inhibition of PI3K-mTOR activity in cetuximab-treated DiFi xenografts.

<p>DiFi tumor xenografts were imaged on day 7 of 20 mg/kg or 40 mg/kg cetuximab treatment regimens. (<b>A</b>) [¹⁸F]-FLT PET was only reduced in DiFi xenografts treated at the 40 mg/kg level (p = 0.0012). (<b>B</b>) Western blot analysis of tissues harvested immediately after imaging confirmed that TK1 protein levels were only decreased at the 40 mg/kg dose level. Increased p27 protein levels were observed at the 40 mg/kg dose levels. Increased p-AKT Ser473 protein levels were observed at the 20 mg/kg dose level. (<b>C</b>) Similar to <i>in vitro</i> observations, <i>TK1</i> mRNA was significantly reduced at both 20 mg/kg (p = 0.0009) and 40 mg/kg (p = 0.0001) dose levels. (<b>D</b>) IHC analysis of p-rpS6 and p-AKT Ser473 showed slightly elevated staining levels at 20 mg/kg. However, both markers were greatly reduced at 40 mg/kg. Ki67 immunoreactivity was reduced at both levels of cetuximab exposure.</p

[¹⁸F]-FLT PET reflects BEZ235-dependent inhibition of PI3K/mTOR activity in PLX4720 treated COLO 205 xenografts.

<p> Xenograft-bearing mice were imaged with [¹⁸F]-FLT PET on treatment day 4. (<b>A</b>) [¹⁸F]-FLT uptake was diminished in the combination treatment cohort relative to vehicle (p = 0.0087), but not single agent PLX4720- or BEZ235-treated cohorts. (<b>B</b>) Western blot of xenograft tissue harvested immediately following imaging illustrated elevated p-ERK and p-rpS6 levels in PLX4720-treated mice. Combining PLX4032 with BEZ235 resulted in reduced p-ERK and p-rpS6 protein levels. (<b>C</b>) TK1 levels, as measured by IHC, were reduced only in the combination treatment group in agreement with [¹⁸F]-FLT PET. (<b>D</b>) Consistent with <i>in vitro</i> studies, diminished TK1 levels, and consequently [¹⁸F]-FLT PET, correlated with elevated p27 that was elevated only in the combination treated group.</p