Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras-driven lung tumours

Abstract

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision-cut slices from Kras-driven non-small-cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen-activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short-term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3-kinase-mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology-associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: non-small-cell lung cancer; oncogenic signalling; precision-cut slices; prostate cancer; spatial heterogeneity; targeted therapy.

Figure 1

Histopathology‐related heterogeneity of KL and KP NSCLC tumours. (A) IHC images depict p63 and NKX2.1 in KL and KP tumours. The solid line indicates mucinous AC, the dotted lines (blue) indicate the ASCs and the AC cores of ASCs (black) or typical necrotic areas in ASC 33. Scale bars: 2 mm (low‐magnification images) and 100 μm (high‐magnification images). (B) Quantification and representative IHC images depicting Ki67 (stained area as percentage of whole tumour area) in KL and KP tumours (four to six analysed mice per tumour group; each dot represents one tumour). Scale bar: 100 μm. One‐way anova (average per mouse was used as an experimental unit). p < 0.0001. Data are shown as mean ± standard deviation.

Figure 2

Oncogenic signalling pathway phosphoproteins show heterogeneous spatial distribution in KL and KP NSCLCs. (A) Quantified expression (percentage of tumour area stained) of pSRC(Y416), pAKT(S473), pERK1/2, pAMPK and p4EBP1 in Ad5‐CC10‐Cre‐induced KL SCC or AC of ASC tumour regions and Ad5‐SPC‐Cre‐induced KL or KP AC tumours. Each dot represents a tumour (11–29 tumours from seven mice for KL ASC, and five mice each for KL and KP AC), and lines and whiskers indicate mean ± standard deviation of the average values of individual tumours per histotype group. Significance was assessed with t‐tests (average per mouse was used as an experimental unit): *p < 0.05, **p < 0.01, ***p < 0.001. (B) IHC images of stained tumours induced by Ad5‐CC10‐Cre (KL) or Ad5‐SPC‐Cre (KL and KP); one image for each of the phosphoproteins and lesion groups is shown by a beeswarm plot in (A). For Ad5‐CC10‐Cre KL tumours, the solid line indicates mucinous AC, and black and red dotted lines define outer SSCs and inner AC cores of ASCs, respectively. Scale bars: 1 mm.

Figure 3

KL and KP tumour slices show cytotoxic responses to combined targeting of the MAPK and PI3K–mTOR pathways. (A) Signalling diagram depicting molecular compounds and their targets utilised in this study. (B) Responses following 24 h of treatment with single compounds: 0.5 or 1 μm dact, 0.5 μm sel, or 1 μm sar. The viable tissue area (percentage of tumour area) in drug‐treated slices is correlated with the viable area in neighbouring DMSO‐treated controls. The diagonal line corresponds to equal viability, and the grey area corresponds to the region with <5% difference between treated and control slices. Representative H&E images of matching DMSO‐treated and drug‐treated slices are shown; regions of cell death are pseudo‐masked in pink, and viable areas in purple. Scale bar: 300 μm. Greek letters (α, β, γ, δ) and arrows in the scatterplot indicate the tumours shown in the images. KL ASC samples represent data measured in larger SCC histotype regions; no visible response was detected in core KL ACs of ASC regions. (C) Correlation of viable tissue area in drug‐treated and matching DMSO control slices (percentage of total tumour area) in KL and KP tumours following 24 h of combination treatment with dact + sar (0.5 or 1 μm + 1 μm), sel + sar (0.5 μm + 1 μm), or dact + sel (0.5 or 1 μm + 0.5 μm). Of these, only the dact + sel combination elicited cytotoxicity, which was quantified for the four histotype groups. (D) Comparison of drug responses after 24 h of dact + sel treatment in the four different histotype groups: decrease in viability (%) measured as the ratio of the viable area in the drug‐treated slice and the matching DMSO control. One‐way anova was used for statistical comparison.

Figure 4

Oncogenic signalling activities at treatment onset correlate with dact + sel combination treatment responses. (A) Drug responses following 24 h of dact + sel treatment in slices representing KL SCC of ASC, KL AC of ASC, KL AC and KP AC tumour tissue, plotted against expression (percentage of tumour area) of p4EBP1 or pERK1/2, indicating targeted pathway activities in source tumours at treatment onset. Viability following drug treatment is depicted as relative decrease in viability (%). Spearman coefficients (ρ) are indicated with significance: *p < 0.05, ***p < 0.001. Greek letters (α, β, γ, δ) indicate tumour samples shown in (C). (B) Scatterplot depicting the correlation of pERK (y‐axis) and p4EBP1 (x‐axis) at treatment onset in relation to the relative decrease in viability (%; balloon size) in dact + sel‐treated slices after 24 h. The four tumour groups are indicated by colours; α, β, γ, δ indicate the tumour samples shown in (C). (C) Selected phosphoprotein expression and drug response data from slice experiments plotted in (A) and (B), indicated by Greek letters (α, β, γ, δ). Shown are representative images of H&E‐stained sections with masks for dead (pink) and viable (purple) tissue, and IHC stains of p4EBP1 and pERK in the corresponding 0‐h slices. Scale bars: 500 μm (top row) and 300 μm (bottom row).

Figure 5

Combination treatment response is determined by spatial distribution of the targeted pathway activities in tumour slices. (A) Correlation of dact + sel treatment with the areas of p4EBP1 and pERK overlap (%) in neighbouring 0‐h slices in the four different tumour groups. Drug response depicted as relative decrease in viability (%) is measured as the ratio of quantified viable area in drug‐treated and corresponding DMSO‐treated controls. Spearman coefficients (ρ) are indicated with significance: *p < 0.05. (B) IHC images depicting p4EBP1 expression (blue), pERK expression (red) or their overlapping expression (green) at 0 h in three KP or KL AC tumours selected from the data plotted in (A) and (B). H&E images of drug‐treated and matching DMSO‐treated controls are pseudo‐marked to indicate dead (pink) and viable (purple) areas. Scale bars: 1 mm (immunohistochemistry) and 200 μm (H&E). (C) H&E images of DMSO‐treated or dact + sel‐treated slices showing dead (pink) and viable (purple) areas of a KL SCC of an ASC tumour selected from the data plotted in (A) and (B). IHC images depicting p4EBP expression (blue), pERK expression (red) or their overlapping expression (green) at treatment onset. The yellow dotted line outlines an area with low cytotoxicity (H&E image), lower pERK expression (IHC image) and lower phosphoprotein overlap than in the area marked by the orange dotted line. Scale bars: 500 μm.