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Figure S1. Additional RPPA endpoints. (PDF 89474 kb)

Figure S1. Metabolites plasma concentrations (MPR and oxo-4-HPR) determined in mice after single oral treatment of 4 doses of Bio-nFeR: 10, 50, 100 and 200 mg/Kg. The analysis was performed after 0, 24, 48 h. Figure S2. Evaluation of the 50% inhibitory concentration (IC50) of BionFeR and NCI-FeR formulation was done on a representative lung cancer stem cell line (LCSC). The cells were exposed to the indicated drug doses (0–30 μM) and cell viability was evaluated by CellTiter-Glo after 72 h. Figure S3. A) Sphingomyelin-Ceramide (SM-CER) and Sphingomyelin- Dihydro-Ceramide (SM-DH-CER) content in lung (LC), melanoma (MEL) and colon (CRC) tumors treated with Bio-nFeR at single 100–150 mg/Kg administration were quantified through Ultra High Performance Liquid Chromatography (UHPLC), normalized to control and plotted as fold change. B) Quantification of Glucosyl-Ceramide (GLC-CER) and Glucosyl-Dihydro-Ceramide (GLC-DH-CER) in the same samples as in A, normalized to control and plotted as fold change. Figure S4. Cancer stem cell marker expression in CSC-culture conditions and in CSC-derived xenografts. A) Flow cytometry analysis of Aldefluor in (left panels) and CD44v6 (middle and right panels) in the indicated CSC and xenograft samples. B) Immunoblot analysis of CSC antigens c-Kit and SOX-2 in the indicated control (−) or BionFeR treated (+) melanoma xenografts. β-actin blot was used for equal loading control. Figure S5. Growth curves of lung CSC-derived xenografts in control mice or mice treated with Bio-nFeR or NCI-FeR at 100 mg/kg dose for the indicated times. Mean ± standard error is shown. *P

Table S1. Complete list of antibodies used for RPPA analysis and their main related information. (PDF 26 kb)

Figure S2. Clonogenic Assay. (PDF 440 kb)

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently, conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However, the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here, we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay, monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly, pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast, brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion, patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions, limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus, CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells, as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells.

Cellule tumorali che mostrano un fenotipo e posseggono proprietà di cellule staminali, sono definite cellule staminali tumorali (CSC). Esse sono state identificate in diversi tumori solidi e si presume che siano le responsabili della crescita del tumore, della chemio-resistenza, della formazione di metastasi e delle recidive osservate nei pazienti e. Una sottopopolazione di cellule con un più alto potenziale staminale, è stata già identificata all’interno dei sarcomi. Queste cellule presentano un elevato potenziale tumorigenico, sono chemioresistenti, esprimono marcatori di tipo embrionale e possiedono un fenotipo di side population (SP). Il leiomiosarcoma (LMS) è un sarcoma dei tessuti molli che origina presumibilmente da cellule indifferenziate di origine mesenchimale, le Cellule Staminali Mesenchimali (MSC). Le frequenti ricadute e la chemioresistenza osservata in pazienti affetti da leiomiosarcoma potrebbe derivare dalla mancata efficacia dei farmaci, attualmente utilizzati in clinica, sulla sottopopolazione di CSC. Ne emerge che terapie innovative che abbiano come bersaglio principale le cellule staminali tumorali (CSC), potrebbero migliorare notevolmente la prognosi dei pazienti affetti da LMS. In questo lavoro abbiamo espanso in vitro cellule staminali di LMS da biopsia asportata da paziente con lo scopo di contrastare questa neoplasia colpendo la popolazione più tumorigenica. CSC di LMS sono state espanse in vitro sia come “ sferoidi” che come “monostrato di cellule aderenti” ottenuto in condizioni utilizzate per coltivare le MSC. Esse presentano un fenotipo di tipo mesenchimale indifferenziato, hanno un’alta percentuale di cellule con fenotipo SP, un’elevata capacità di espellere farmaci, un illimitato potenziale proliferativo e di auto autoreplicazione; inoltre sono in grado di differenziare nei diversi lineage di tipo mesenchimale. Le CSC di LMS sono chemioresistenti, altamente tumorigeniche, e riproducono fedelmente il tumore del paziente in topi immunodeficenti. Esse presentano attivazione del pathway di EGFR/AKT/MAPK, suggerendo la possibilità di inibire la loro chemioresistenza mediante il blocco di questa via di segnale. Il trattamento combinato con IRESSA più Vincristina determina l’inattivazione della via del segnale di EGFR, riduce la SP, esercita un’elevata citotossicità in vitro e una forte attività antitumorale in xenografts generati dalle CSC di LMS, colpendo sia le cellule staminali sia differenziate. In conclusione, il trattamento con l’inibitore di EGFR in combinazione con la vincristina determina un marcato effetto antitumorale sia in vitro che in vivo contro le cellule di LMS, fornendo una possibile terapia antitumorale per pazienti affetti da LMS. This work was supported by the Italian Association for Cancer Research and the Italian Health Ministry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary Methods. Figure S1. RAF inhibition induces BRAF/CRAF heterodimerization in KRAS-mut contexts. Figure S2. Effects of trametinib, dabrafenib and their combination on cell growth and cell cycle distribution in A549 and MiaPaCa2 cells. Figure S3. Effects of trametinib and dabrafenib combination in lung cancer cells. Figure S4. Effects of trametinib and dabrafenib combination in pancreatic cancer cells. Figure S5. Effects of trametinib, dabrafenib and their combination in LCSC. Figure S6. Statistical correlation between KRAS status and pharmacological interactions. Figure S7. Effects of lapatinib in EGFR/HER2 amplified lung cancer cell lines. Table S1. Summary of the genetic status of the cell lines analyzed and response to treatments. (PDF 2519 kb)

Supplementary Methods. Figure S1. RAF inhibition induces BRAF/CRAF heterodimerization in KRAS-mut contexts. Figure S2. Effects of trametinib, dabrafenib and their combination on cell growth and cell cycle distribution in A549 and MiaPaCa2 cells. Figure S3. Effects of trametinib and dabrafenib combination in lung cancer cells. Figure S4. Effects of trametinib and dabrafenib combination in pancreatic cancer cells. Figure S5. Effects of trametinib, dabrafenib and their combination in LCSC. Figure S6. Statistical correlation between KRAS status and pharmacological interactions. Figure S7. Effects of lapatinib in EGFR/HER2 amplified lung cancer cell lines. Table S1. Summary of the genetic status of the cell lines analyzed and response to treatments. (PDF 2519 kb)