Protocol to utilize fresh uncultured human lung tumor cells for personalized functional diagnostics

Peer reviewe

Functional diagnostics using fresh uncultured lung tumor cells to guide personalized treatments

Functional profiling of a cancer patient's tumor cells holds potential to tailor personalized cancer treatment. Here, we report the utility of fresh uncultured tumor-derived EpCAM(+) epithelial cells (FUTCs) for ex vivo drug response interrogation. Analysis of murine Kras mutant FUTCs demonstrates pharmacological and adaptive signaling profiles comparable to subtype-matched cultured cells. By applying FUTC profiling on non-small cell lung cancer patient samples, we report robust drug-response data in 19 of 20 cases, with cells exhibiting targeted drug sensitivities corresponding to their oncogenic drivers. In one of these cases, an EGFR mutant lung adenocarcinoma patient refractory to osimertinib, FUTC profiling is used to guide compassionate treatment. FUTC profiling identifies selective sensitivity to disulfiram and the combination of carboplatin plus etoposide, and the patient receives substantial clinical benefit from treatment with these agents. We conclude that FUTC profiling provides a robust, rapid, and actionable assessment of personalized cancer treatment options.Peer reviewe

Protocol to utilize fresh uncultured human lung tumor cells for personalized functional diagnostics

Drug sensitivity data acquired from solid tumor-derived cultures are often unsuitable for personalized treatment guidance due to the lengthy turnaround time. Here, we present a protocol for determining ex vivo drug sensitivities using fresh uncultured human lung tumor-derived EpCAM+ epithelial cells (FUTCs). We describe steps for drug testing in FUTCs to identify tumor cell-selective single or combination therapy in 72 h of sample processing. The FUTC-based approach can also be used to predict in vivo resistance to known targeted therapies. For complete details on the use and execution of this protocol, please refer to Talwelkar et al. (2021).</p

Receptor Tyrosine Kinase Signaling Networks Define Sensitivity to ERBB Inhibition and Stratify Kras Mutant Lung Cancers

Most non-small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53, or STK11/LKB1 alterations. By coupling ex viva drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from Kras(G12D/+);Lkb1(fl/fl) (KL) tumors or AC cultures from Kras(G12D/+);p53(fl/fl) (KP) tumors. Although p53-null cells readily propagated as conventional cultures, Lkb1-null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC niltures. Furthermore, pan-ERBB inhibition reduced the clonogenidty of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.Peer reviewe

Inhibiting cycloxygenase and ornithine decarboxylase by diclofenac and alpha-difluoromethylornithine blocks cutaneous SCCs by targeting Akt-ERK axis.

Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser(473) & thr(308)) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion

DFMO+diclofenac decrease the expression levels of ODC and COX-2 through Akt and ERK signaling axis.

<p>(A) Western blot analysis showing expression of ODC and COX-2. The bar diagram represents relative expression levels of these proteins (ODC: @ = 0.04, # = NS,  = 0.01, & = NS, ? = 0.02; COX-2: @ = NS, # = 0.04,  = 0.008, & = 0.003 ? = NS). No significant changes were noted in the levels of COX-1 (data not shown) upon DFMO or diclofenac treatments or both in these tumors. (B) DFMO+diclofenac treatment exert some target-unrelated effects. Individual and combinatorial treatments of DFMO and diclofenac on the levels of p-ERK (@ = 0.05, # = 0.03,  = 0.001, & = 0.002 and ? = 0.05) and p-MAPKAP-2 (@ = NS, # = 0.03,  = 0.008, & = 0.04 and ? = 0.05), an important mediators of tumor growth. (C) Effect of DFMO and diclofenac treatments on the levels of p-Akt ser 473 (@ = NS, # = 0.05,  = 0.02, & = 0.005 and ? = 0.005) and thr 308 (@ = NS, # = 0.05,  = 0.02, & = 0.03 and ? = 0.04) when treated alone and in combination. @ - significant when DFMO alone compared to vehicle-treated control, # - significant when diclofenac alone compared to vehicle-treated control, $ - significant when DFMO+diclofenac compared to vehicle, & and ? - significant when DFMO+diclofenac compared to single treatment of DFMO and diclofenac respectively.</p

Akt over-expression resists the effect of DFMO+diclofenac.

<p>Treatment of myr-flag-Akt overexpressing cells with DFMO and diclofenac resisted the changes in the levels of p-Akt ^ser-473 and cyclin D1 as compared to empty vector control and wild type A431 cells.</p

DFMO+diclofenac decreases proliferation and induce cell death by apoptosis in human epidermoid xenografts.

<p>(A) Tissue sections from A431 xenograft tumors were stained for PCNA or TUNEL. DFMO+diclofenac treatment on expression levels of PCNA and TUNEL-positive apoptotic cells as compared to vehicle and their individual treatments. (B) Western blot analysis was done by randomly selecting two individual samples from each group. The effect of ODC and COX-2 inhibitors treated as single agents and in combination on the expression of cyclin D1 and apoptotic marker proteins. The bar diagram represents relative expression level of these proteins & the error bars demonstrate the standard error between two individual samples selected from each group. (cyclin D1: @ = NS, # = 0.04,  = 0.001, & = 0.01, ? = 0.05; Bcl-2: @ = NS, # = 0.04,  = 0.01, & = 0.05, ? = 0.05; cleaved caspase-3: @ = 0.005, # = 0.007,  = 0.002, & = 0.004 ? = NS). @ - significant when DFMO alone compared to vehicle-treated control, #- significant when diclofenac alone compared to vehicle-treated control, - significant when DFMO+diclofenac compared to vehicle, & and ?-significant when DFMO+diclofenac compared to single treatment of DFMO and diclofenac respectively. Identical β-actin loading controls are denoted by symbol ‘†’ ‘¥’ ‘‡’ and ‘€’ in various figures.</p

Flow diagram showing effects of ODC and COX-2 combined inhibition on the molecular targets involved in human SCC pathogenesis.

<p>ODC and COX-2 over-expression leads to increase in polyamine biosynthesis and PGE2 levels respectively, activating Akt and ERK-dependent signaling pathways which modulate tumor growth and invasion through enhanced EMT resulting in aggressive growth of SCCs.</p