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.

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

“…This indicates that it is important to investigate whether the targeted in situ biological functions are appropriately preserved during cultivation, prior to perturbation studies. Despite these findings, we showed that tumor slices can model spatial response to targeted therapies, midst drug treatments remain brief (24 h) and are initiated at the onset of culturing 26 . The following protocol describes important validation aspects relevant to the establishment and analysis of tumor slice cultures, prior to their application in pharmacological drug testing.…”

“…2. Calculate the relative viability of the slices cultivated for different time points compared to the nearest 0 h slice as done in our previous study (Närhi et al, Figure S2B and C) 26 . Similarly, assess potential intra-slice viability gradients by quantifying viability in the top, middle and bottom section of a cultured slice, and calculate the relative viability of each section to its closest 0 h slice.…”

“…Using slices derived from breast, prostate and lung cancer models, this joint effort utilized qualitative read-outs as well as quantitative hematoxylin and eosin (H&E) -based readouts to demonstrate a requirement for atmospheric oxygen and stagnant filter supports to sustain the viability of cultured slices until 72 h. Furthermore, immunohistochemistry (IHC) analyses on cultured slices revealed intra-slice viability gradients, evidenced as necrosis gradients in slices derived from murine non-small cell lung cancer (NSCLC), estrogen receptor (ER), HIF1α and γH2AX gradients in breast cancer slices, or androgen receptor (AR) expression gradients in prostate cancer slices 25 . Interestingly, intra-slice viability gradients in 24 h cultures of murine NSCLC were rescued by cultivation in a rotating incubation unit, and our recent study showed that viability was extended to 72 h 26 . Particularly the top side remained most viable 26 , endorsing that drug response analyses on slices are best carried out on this side of the tissue.…”

“…Interestingly, intra-slice viability gradients in 24 h cultures of murine NSCLC were rescued by cultivation in a rotating incubation unit, and our recent study showed that viability was extended to 72 h 26 . Particularly the top side remained most viable 26 , endorsing that drug response analyses on slices are best carried out on this side of the tissue.…”

“…Even though it remains a question as to how far tissue slices can recapitulate in situ tumor functions, they have been extensively used to test responses to anti-cancer agents, including targeted drugs, monoclonal antibodies and chemotherapy agents 10,11,13,14,18,27 . We recently showed that murine NSCLC slices show dynamic changes in proliferation and oncogenic signaling activities following cultivation when compared to freshly cut 0 h slices 26 . This indicates that it is important to investigate whether the targeted in situ biological functions are appropriately preserved during cultivation, prior to perturbation studies.…”