Storage in anhydrous ethanol at -20°C preserves immunofluorescence signals from cytokeratin 7 and DNA.

A., Images for control patient 4844 capture typical variability observed with immunofluorescence. Red, CK7, cyan, DNA. B., kinetics of overall stack intensities, as measured by the center-of-mass of the pixel intensity histogram, neither significantly decreased nor increased over the a two-month period when tissue sections were maintained dehydrated and at freezing temperatures. (PDF)</p

Cell volumes of each KRAS+ patient as a function of ploidy.

"-", " = ", "+", classifications based on cells with sub-, green, proportional, blue, or supraproportional, red, behavior, used to construct Fig 3B. See Methods and S1 Data tab E for classification regime. Each dot represents a single cell measurement (n = 4082, related to S1 Data, tab B). Rows, stages 1–3. Some outliers are excluded from the edges of the plot for clarity and their data can be found in S1 Data, tab B. (PDF)</p

Polyploid and euploid N:C ratios binned over all stages.

Top, Normal AT2 cells (n = 802). All nuclei, N = 21, n = 4078. EGFR+, all ploidies, N = 10, n = 2194. KRAS+, all ploidies, N = 11, n = 1884. Euploids, N:C ratios from cells with estimated ploidies of from 1.6–4.4n (n = 2668). EGFR euploids, n = 1411. KRAS euploids, n = 1257. Fits are a Gaussian mixture model, top, and lognormal, other histograms. Small Gaussians overlaid are the first Gaussian from the mixture model fit to normal AT2 cells, corresponding to the 2n population. Goodness-of-fit statistics available in S1 Data, tab G. (PDF)</p

Lamin A+C envelope wrinkling is only weakly associated with nuclear volume.

Related to S26 Fig, plots of the volume of the surface segmentation of Lamin A+C staining as a function of the sphericity reported by Imaris software, binned by genotype. Left, EGFR+, right, KRAS+. Lamin A+C Inner Volume = the volume of the envelope built from the Lamin A+C channel, which is significantly smaller than the volume of the nucleus as calculated from DNA thresholding (S5 Fig and Methods). Both large and small nuclei display a range of sphericities, which is not consistent with rounding of nuclei due to nuclear swelling. (PDF)</p

Behavior of AT2 process networks at various scales in human normal and tumor lung tissue and relation to cell-cell junctions.

A., zoom views of proSPC, green, and CK7, red, showing how process networks follow the network of intracellular cytokeratin without displaying consistent colocalization (yellow in merge, relative to green in merge). This behavior may be expected in the case of multilamellar vesicles, structures which explain the puncta observed in AT2 cell bodies. White arrows, the distinctive pathology of AT2 processes in tumor cells is a progressive depletion of multilamellar bodies (puncta) and enhancement at cell-cell junctions. Neighbors often display a gradient in vesicle expression, from cells that appear AT2-like, to those with weak process staining and aberrant-appearing paths (see also, S4 and S5 Videos). Scale, 5 μm. B., colocalization of proSPC, green, and E-cadherin, red, is partial at the cell boundary, but “subnetworks” and what may be terminating branches are apparent (right image, darker regions). The “loop” that is visible is typical of the shape of large, flat, AT1 cell bodies in these alveoli. Scale, 20 μm. (PDF)</p

Cell-enlargement of near-euploid epithelia cells is a signature feature of resected lung adenocarcinoma tumors that can be detected using the side-scattering parameter in flow-cytometry.

Related to Fig 5. CD45-/Zombie Red-/EPCAM+/Near-euploid cells gently dissociated from normal, top, or tumor, bottom surgical resections were further split according to side-scattering width (SSC-W). SSC-W gates: 1 = 64–79, 2 = 84–99, 3 = 101–113. Bars represent the fraction of all cells in gates G1-G4 for SSC-W vs SSC-H (See Fig 5B). Gate G4 for SSC-W is not shown, and contains sparing aggregates and debris. Right plot, mean of all fractions found in G3, containing the largest singlet cells. Error bars, SEM. (PDF)</p

Cell volumes of each EGFR+ patient as a function of ploidy.

"-", " = ", "+", classifications based on cells with sub-, green, proportional, blue, or supraproportional, red, behavior, used to construct Fig 3B. See Methods and S1 Data tab E for classification regime. Each dot represents a single cell measurement (n = 4082, related to S1 Data, tab B). Rows, stages 1–3. Some outliers are excluded from the edges of the plot for clarity and their data can be found in S1 Data, tab B. (PDF)</p

S11 Fig -

A., Polyploid and B., euploid N:C ratios by stage and genotype. Small Gaussian fits, first 2n population from a mixture-model fit to normal AT2 cell nuclei and cell bodies. Fits, top row, two Gaussian mixture, other fits, lognormal. Goodness-of-fit statistics and parameters from regression fit to normal AT2 cells as well as sample sizes by stage and genotype available in S1 Data, tab G. (PDF)</p

SSC-A vs FSC-A plots for samples at various stages of our protocol to sort tumor cells by size.

Parent, the digest stained before MACS CD45+ depletion. Plunge, the column retentate containing CD45+ and some CD45- cells. Flow-through, CD45- cells. Zombie Red -, EPCAM+, Euploids, related to the corresponding gates shown in Fig 5A left-to-right. G1-G3, size gates for SSC-W, related to Fig 5B, show the expected increase in FSC-A and SSC-A with increasing cell size occurs, but data resolution and range is not optimal. (PDF)</p

Only a handful of examples of flat-looking tumor cells were observed.

A., z-projections at 63X magnification of flat-looking cells are still many times the thickness of an AT1 cell, suggesting if any developmental programme remains for flattening, it is altered from the wild-type. B., corresponding cell models. Scale, 20 μm. (PDF)</p