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

Peer reviewe

Development of an adenosquamous carcinoma histopathology - selective lung metastasis model

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

Precision medicine for lung cancer : models, functional assays and mechanisms

The introduction of targeted therapies and immune checkpoint inhibitors has significantly improved outcomes for a subset of non-small cell lung cancer (NSCLC) patients. These treatments, however, are never curative as clinical resistance typically develops over time, resulting in disease recurrence and patient’s death. Furthermore, not all NSCLC patients can be matched with appropriate targeted or immune-based therapies. Therefore, to overcome drug resistance and provide therapeutic benefit to a larger NSCLC patient population, new therapeutic strategies are urgently needed. Functional profiling of tumor cells collected from cancer patients has the potential to tailor individualized cancer treatments. To assess the translational potential of this strategy for NSCLC, drug sensitivity and resistance testing were conducted using tumor-derived fresh uncultured (passage 0) or cultured (passage 4-10) cells from mouse models and clinical samples. The first part of this thesis concentrated on the utilization of cultures established from murine Kras mutant NSCLC tumors for pharmacological exploration. Drug sensitivity and resistance testing, in conjunction with biochemical dissection of treatment-adaptive RTK activity, led to the functional stratification of Kras mutant NSCLC, and the identification of subtype-selective treatments. The second study in this thesis demonstrated that NSCLC cells obtained from patients can be employed as a reliable pre-clinical model for the identification of single and combinatorial treatment strategies. By employing patient-derived ALK-rearranged NSCLC cells for drug screening, this study discovered PI3Kβ as a potential therapeutic target to improve ALK inhibitor sensitivity. The final section of this thesis assesses the utility of Fresh Uncultured Tumor-derived EpCAM+ Cells (FUTCs) for ex vivo drug response interrogation. The findings of this study showed that patient-derived FUTCs can be reliably utilized for ex vivo drug testing, and thus offering a promising functional diagnostic tool to support individualized cancer treatments. Overall, the results presented in this thesis demonstrate the clinical feasibility and utility of tumor-derived fresh uncultured or cultured cells for pharmacological research: primary cultures can be utilized to examine functional heterogeneity, understand drug resistance mechanisms, and identify combinatorial treatments to improve patient outcomes, whereas FUTC-based functional diagnostic test can be used to identify individualized cancer treatments.Kohdennettujen hoitojen ja immuunitarkastuspisteen estäjien käyttöönotto on parantanut merkittävästi tuloksia osalla ei-pienisoluista keuhkosyöpää (NSCLC) sairastavilla potilailla. Nämä hoidot eivät kuitenkaan ole koskaan parantavia, koska kliininen vastustuskyky kehittyy tyypillisesti ajan myötä, mikä johtaa taudin uusiutumiseen ja potilaan kuolemaan. Lisäksi kaikkia NSCLC-potilaita ei voida yhdistää niille sopiville kohdennetuille lääkkeille tai immuunihoidoille. Siksi uusia terapeuttisia strategioita tarvitaan kiireesti lääkeresistenssin voittamiseksi ja terapeuttisen hyödyn tarjoamiseksi suuremmalle NSCLC-potilasjoukolle. Syöpäpotilailta kerättyjen kasvainsolujen toiminnallisella profiloinnilla on madollista räätälöidä yksilöllisiä syöpähoitoja. Tämän strategian translaatiopotentiaalin arvioimiseksi NSCLC potilaille, lääkeherkkyys- ja resistenssitestit suoritettiin käyttämällä kasvainperäisiä tuoreita viljelemättömiä (siirrostus 0) tai viljeltyjä (siirrostus 4-10) soluja NSCLC-hiirimalleista ja kliinisistä näytteistä. Tämän väitöskirjatyön ensimmäinen osa keskittyi hiiren Kras-mutanteista NSCLC-kasvaimista saatujen viljelmien hyödyntämiseen farmakologisessa tutkimuksessa. Lääkeherkkyys- ja resistenssitestaus yhdessä hoitoadaptiivisen ja biokemikaalisesti dissektoidun RTK-aktiivisuuden kanssa johti toiminnalliseen kerroksellisuuteen ja alityyppiselektiivisten hoitojen tunnistamiseen. Tämän väitöskirjatyön toisen tutkimuksen tulosten mukaan, potilasperäisiä NSCLC-soluja voidaan käyttää luotettavana prekliinisenä mallina lääkekehityksessä. PI3Kβ todettiin olevan mahdollinen terapeuttinen kohde ALK inhibiittorien vasteen nostamisessa, kun potilasperäiset ALK-uudelleenjärjestetyt NSCLC-solut applikoitiin farmakologisiin seulontoihin. Väitöskirjatyön viimeisessä osassa arvioidaan tuoreiden viljelemättömien kasvainperäisten EpCAM+ -solujen (FUTC) käyttökelpoisuutta ex vivo lääkevasteen selvittämisessä. Tämän tutkimuksen tulokset osoittivat, että kasvainperäisiä FUTC-soluja voidaan luotettavasti käyttää ex vivo -lääketestaukseen, mikä tarjoaa lupaavan henkilökohtaisen toiminnallisen diagnostisen työkalun yksilöllisten syöpähoitojen tukemiseen. Kaiken kaikkiaan tässä väitöskirjassa esitetyt tulokset osoittavat kasvainperäisten tuoreiden viljelemättömien tai viljeltyjen solujen kliinisen toteutettavuuden ja käyttökelpoisuuden farmakologiseen tutkimukseen: primääriviljelmillä voidaan tutkia toiminnallista heterogeenisyyttä, ymmärtää lääkeresistenssimekanismeja ja tunnistaa kombinatorisia hoitoja potilastulosten parantamiseksi, kun taas FUTC-pohjaista toiminnallista diagnostista analyysia voidaan käyttää yksilöllisten syöpähoitojen tunnistamiseen

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

Inactivation of AMPK leads to attenuation of antigen presentation and immune evasion in lung adenocarcinoma

International audiencePurpose: Mutations in STK11 (LKB1) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TIME) and resistance to immunotherapy. The mechanisms underpinning the establishment and maintenance of a cold TIME in LKB1-mutant LUAD remain poorly understood. In this study, we investigated the role of the LKB1 substrate AMPK in immune evasion in human non-small cell lung cancer (NSCLC) and mouse models and explored the mechanisms involved.Experimental design: We addressed the role of AMPK in immune evasion in NSCLC by correlating AMPK phosphorylation and immune-suppressive signatures and by deleting AMPKα1 (Prkaa1) and AMPKα2 (Prkaa2) in a KrasG12D -driven LUAD. Furthermore, we dissected the molecular mechanisms involved in immune evasion by comparing gene-expression signatures, AMPK activity, and immune infiltration in mouse and human LUAD and gain or loss-of-function experiments with LKB1- or AMPK-deficient cell lines.Results: Inactivation of both AMPKα1 and AMPKα2 together with Kras activation accelerated tumorigenesis and led to tumors with reduced infiltration of CD8+/CD4+ T cells and gene signatures associated with a suppressive TIME. These signatures recapitulate those in Lkb1-deleted murine LUAD and in LKB1-deficient human NSCLC. Interestingly, a similar signature is noted in human NSCLC with low AMPK activity. In mechanistic studies, we find that compromised LKB1 and AMPK activity leads to attenuated antigen presentation in both LUAD mouse models and human NSCLC.Conclusions: The results provide evidence that the immune evasion noted in LKB1-inactivated lung cancer is due to subsequent inactivation of AMPK and attenuation of antigen presentation

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