Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness.

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Chemotherapy-induced neutropenia elicits metastasis formation in mice by promoting proliferation of disseminated tumor cells.

Chemotherapy is the standard of care for most malignancies. Its tumor debulking effect in adjuvant or neoadjuvant settings is unquestionable, although secondary effects have been reported that paradoxically promote metastasis. Chemotherapy affects the hematopoietic precursors leading to myelosuppression, with neutropenia being the main hematological toxicity induced by cytotoxic therapy. We used renal and lung murine tumor models metastatic to the lung to study chemotherapy-induced neutropenia (CIN) in the metastatic process. Cyclophosphamide and doxorubicin, two myelosuppressive drugs, but not cisplatin, increased the burden of artificial metastases to the lung, by reducing neutrophils. This effect was recapitulated by treatment with anti-Ly6G, the selective antibody-mediated neutrophil depletion that unleashed the formation of lung metastases in both artificial and spontaneous metastasis settings. The increased cancer dissemination was reversed by granulocyte-colony stimulating factor-mediated boosting of neutrophils in combination with chemotherapy. CIN affected the early metastatic colonization of the lung, quite likely promoting the proliferation of tumor cells extravasated into the lung at 24-72 hours. CIN did not affect the late events of the metastatic process, with established metastasis to the lung, nor was there any effect on the release of cancer cells from the primary, whose growth was, in fact, somewhat inhibited. This work suggests a role of neutrophils associated to a common cancer treatment side effect and claims a deep dive into the relationship between chemotherapy-induced neutropenia and metastasis.

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth.

BACKGROUND: The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches. METHODS AND RESULTS: Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5ß1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models. CONCLUSIONS: This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.

The PARP1 Inhibitor AZD5305 Impairs Ovarian Adenocarcinoma Progression and Visceral Metastases in Patient-derived Xenografts Alone and in Combination with Carboplatin.

PARP inhibitors (PARPi) have changed the management of patients with ovarian cancer and their effectiveness has been demonstrated especially in homologous recombination repair-deficient tumors. These first-generation drugs target PARP1, but also PARP2 and other family members potentially responsible for adverse effects that limit their therapeutic potential and restrict their use in combination with chemotherapeutic agents. We investigated ovarian cancer patient-derived xenografts (OC-PDXs) to assess whether malignant progression could be impaired by a novel inhibitor selective for PARP1 (AZD5305) and to assess the potential of its combination with carboplatin (CPT), the standard-of-care for patients with ovarian cancer. In BRCA-mutated OC-PDXs, AZD5305 achieved greater tumor regressions and longer duration of response as well as a superior impairment of visceral metastasis and improved survival benefit compared with the first-generation dual PARP1/2 inhibitors. The combination of AZD5305 plus CPT was more efficacious than single agents. Subcutaneously growing tumors experienced regression that persisted after therapy stopped. Combination efficacy was greater against tumors that did not respond well to platinum, even at a dose at which AZD5305 monotherapy was ineffective. The combination therapy impaired metastatic dissemination and significantly prolonged the lifespan of mice bearing OC-PDXs in their abdomen. This combination benefit was evident even when CPT was used at suboptimal doses, and was superior to full-dose platinum treatment. These preclinical studies demonstrate that the PARP1-selective inhibitor AZD5305 retains and improves the therapeutic benefit of the first-generation PARPi, providing an opportunity to maximize benefits for this class of anticancer agents. Significance: Selective PARP1i AZD5305 can exceed the efficacy of first-generation PARPi, which target both PARP1 and PARP2, and potentiates the efficacy of CPT when given in combination. AZD5305 alone or in combination with platinum delayed visceral metastasis, ultimately extending the lifespan of OC-PDX-bearing mice. These preclinical models mimic the progression of the disease occurring in patients after debulking surgery, and are translationally relevant.

Endoplasmic reticulum oxidoreductin 1-alpha deficiency and activation of protein translation synergistically impair breast tumour resilience.

BACKGROUND AND PURPOSE: Endoplasmic reticulum (ER) stress triggers an adaptive response in tumours which fosters cell survival and resilience to stress. Activation of the ER stress response, through its PERK branch, promotes phosphorylation of the α-subunit of the translation initiation factor eIF2, thereby repressing general protein translation and augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase, ERO1-alpha EXPERIMENTAL APPROACH: Here, we show that ISRIB, a small molecule that inhibits the action of phosphorylated eIF2alpha, activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1-alpha, enfeebling breast tumour growth and spread. KEY RESULTS: ISRIB represses the CHOP signal, but does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a marked perturbing effect on ERO1-deficient triple-negative breast cancer cells, which display impaired proteostasis and have adapted to a low client protein load in hypoxia, and ERO1 deficiency impairs VEGF-dependent angiogenesis. ERO1-deficient triple-negative breast cancer xenografts have an augmented ER stress response and its PERK branch. ISRIB acts synergistically with ERO1 deficiency, inhibiting the growth of triple-negative breast cancer xenografts by impairing proliferation and angiogenesis. CONCLUSION AND IMPLICATIONS: These results demonstrate that ISRIB together with ERO1 deficiency synergistically shatter the PERK-dependent adaptive ER stress response, by restarting protein synthesis in the setting of impaired proteostasis, finally promoting tumour cytotoxicity. Our findings suggest two surprising features in breast tumours: ERO1 is not regulated via CHOP under hypoxic conditions, and ISRIB offers a therapeutic option to efficiently inhibit tumour progression in conditions of impaired proteostasis.

Integrated molecular profiling of patient-derived ovarian cancer models identifies clinically relevant signatures and tumor vulnerabilities.

High-grade serous ovarian carcinoma (HGSOC) is a highly aggressive and intractable neoplasm, mainly because of its rapid dissemination into the abdominal cavity, a process that is favored by tumor-associated peritoneal ascites. The precise molecular alterations involved in HGSOC onset and progression remain largely unknown due to the high biological and genetic heterogeneity of this tumor. We established a set of different tumor samples (termed the As11-set) derived from a single HGSOC patient, consisting of peritoneal ascites, primary tumor cells, ovarian cancer stem cells (OCSC) and serially propagated tumor xenografts. The As11-set was subjected to an integrated RNA-seq and DNA-seq analysis which unveiled molecular alterations that marked the different types of samples. Our profiling strategy yielded a panel of signatures relevant in HGSOC and in OCSC biology. When such signatures were used to interrogate the TCGA dataset from HGSOC patients, they exhibited prognostic and predictive power. The molecular alterations also identified potential vulnerabilities associated with OCSC, which were then tested functionally in stemness-related assays. As a proof of concept, we defined PI3K signaling as a novel druggable target in OCSC.

The DNA-PK Inhibitor AZD7648 Sensitizes Patient-Derived Ovarian Cancer Xenografts to Pegylated Liposomal Doxorubicin and Olaparib Preventing Abdominal Metastases.

Ovarian cancer is the deadliest gynecologic cancer, with a 5-year survival rate of 30%, when the disease has spread throughout the peritoneal cavity. We investigated the efficacy to delay disease progression by the DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648, administered in combination with two of the therapeutic options for patient management: either pegylated liposomal doxorubicin (PLD) or the PARP inhibitor olaparib. Patient-derived ovarian cancer xenografts (OC-PDX) were transplanted subcutaneously to evaluate the effect of treatment on tumor growth, or orthotopically in the peritoneal cavity to evaluate the effect on metastatic spread. AZD7648 was administered orally in combination with PLD (dosed intravenously) or with olaparib (orally). To prove the inhibition of DNA-PK in the tumors, we measured pDNA-PKcs, pRPA32, and γH2AX, biomarkers of DNA-PK activity. AZD7648 enhanced the therapeutic efficacy of PLD in all the OC-PDXs tested, regardless of their BRCA status or sensitivity to cisplatin or PLD. The treatment caused disease stabilization, which persisted despite therapy discontinuation for tumors growing subcutaneously, and significantly impaired the abdominal metastatic dissemination, prolonging the lifespan of mice implanted orthotopically. AZD7648 potentiated the efficacy of olaparib in BRCA-deficient OC-PDXs but did not sensitize BRCA-proficient OC-PDXs to olaparib, despite an equivalent inhibition of DNA-PK, suggesting the need of a preexisting olaparib activity to benefit from the addition of AZD7648. This work suggests that AZD7648, an inhibitor of DNA-PK, dosed in combination with PLD or olaparib is an exciting therapeutic option that could benefit patients with ovarian cancer and should be explored in clinical trials.

PGC1α/ß Expression Predicts Therapeutic Response to Oxidative Phosphorylation Inhibition in Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1ß. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1ß (PGC1α/ß) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/ß-expressing OC-PDX-bearing mice. Conversely, low PGC1α/ß OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/ß expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and ß as biomarkers to refine the selection of patients likely to benefit most from this therapy. SIGNIFICANCE: OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/ß expression and offers an effective approach to manage patients on the basis of PGC1α/ß expression.

VEGF pathway inhibition potentiates PARP inhibitor efficacy in ovarian cancer independent of BRCA status.

Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.

L1CAM promotes ovarian cancer stemness and tumor initiation via FGFR1/SRC/STAT3 signaling.

BACKGROUND: Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. METHODS: The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. RESULTS: We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. CONCLUSIONS: Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

Tumor vascular remodeling by thrombospondin-1 enhances drug delivery and antineoplastic activity.

The disorganized and inefficient tumor vasculature is a major obstacle to the delivery and efficacy of antineoplastic treatments. Antiangiogenic agents can normalize the tumor vessels, improving vessel function and boosting the distribution and activity of chemotherapy. The type III repeats (T3R) domain of thrombospondin-1 contains different potential antiangiogenic sequences. We therefore hypothesized that it might affect the tumor vasculature. Ectopic expression of the T3R domain by the tumor cells or by the host, or administration of recombinant T3R, delayed the in vivo growth of experimental tumors. Tumors presented marked reorganization of the vasculature, with abundant but smaller vessels, associated with substantially less necrosis. Mechanistically, the use of truncated forms of the domain, containing different active sequences, pointed to the FGF2/FGFR/ERK axis as a target for T3R activity. Along with reduced necrosis, the expression of T3R promoted tumor distribution of chemotherapy (paclitaxel), with a higher drug concentration and more homogeneous distribution, as assessed by HPLC and MALDI imaging mass spectrometry. T3R-expressing tumors were more responsive to paclitaxel and cisplatin. This study shows that together with its known role as a canonical inhibitor of angiogenesis, thrombospondin-1 can also remodel tumor blood vessels, affecting the morphological and functional properties of the tumor vasculature. The ability of T3R to reduce tumor growth and improve the response to chemotherapy opens new perspectives for therapeutic strategies based on T3R to be used in combination therapies.

The ER stress response mediator ERO1 triggers cancer metastasis by favoring the angiogenic switch in hypoxic conditions.

Solid tumors are often characterized by a hypoxic microenvironment which contributes, through the hypoxia-inducible factor HIF-1, to the invasion-metastasis cascade. Endoplasmic reticulum (ER) stress also leads tumor cells to thrive and spread by inducing a transcriptional and translational program, the Unfolded Protein Response (UPR), aimed at restoring ER homeostasis. We studied ERO1 alpha (henceforth ERO1), a protein disulfide oxidase with the tumor-relevant characteristic of being positively regulated by both ER stress and hypoxia. Analysis of the redox secretome indicated that pro-angiogenic HIF-1 targets, were blunted in ERO1-devoid breast cancer cells under hypoxic conditions. ERO1 deficiency reduced tumor cell migration and lung metastases by impinging on tumor angiogenesis, negatively regulating the upstream ATF4/CHOP branch of the UPR and selectively impeding oxidative folding of angiogenic factors, among which VEGF-A. Thus, ERO1 deficiency acted synergistically with the otherwise feeble curative effects of anti-angiogenic therapy in aggressive breast cancer murine models and it might be exploited to treat cancers with pathological HIF-1-dependent angiogenesis. Furthermore, ERO1 levels are higher in the more aggressive basal breast tumors and correlate inversely with the disease- and metastasis-free interval of breast cancer patients. Thus, taking advantage of our in vitro data on ERO1-regulated gene products we identified a gene set associated with ERO1 expression in basal tumors and related to UPR, hypoxia, and angiogenesis, whose levels might be investigated in patients as a hallmark of tumor aggressiveness and orient those with lower levels toward an effective anti-angiogenic therapy.

A methodological approach to correlate tumor heterogeneity with drug distribution profile in mass spectrometry imaging data.

BACKGROUND: Drug mass spectrometry imaging (MSI) data contain knowledge about drug and several other molecular ions present in a biological sample. However, a proper approach to fully explore the potential of such type of data is still missing. Therefore, a computational pipeline that combines different spatial and non-spatial methods is proposed to link the observed drug distribution profile with tumor heterogeneity in solid tumor. Our data analysis steps include pre-processing of MSI data, cluster analysis, drug local indicators of spatial association (LISA) map, and ions selection. RESULTS: The number of clusters identified from different tumor tissues. The spatial homogeneity of the individual cluster was measured using a modified version of our drug homogeneity method. The clustered image and drug LISA map were simultaneously analyzed to link identified clusters with observed drug distribution profile. Finally, ions selection was performed using the spatially aware method. CONCLUSIONS: In this paper, we have shown an approach to correlate the drug distribution with spatial heterogeneity in untargeted MSI data. Our approach is freely available in an R package 'CorrDrugTumorMSI'.

Trabectedin and Lurbinectedin Extend Survival of Mice Bearing C26 Colon Adenocarcinoma, without Affecting Tumor Growth or Cachexia.

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBPß/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1ß in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Impact of ERCC1, XPF and DNA Polymerase ß Expression on Platinum Response in Patient-Derived Ovarian Cancer Xenografts.

Platinum resistance is an unmet medical need in ovarian carcinoma. Molecular biomarkers to predict the response to platinum-based therapy could allow patient stratification and alternative therapeutic strategies early in clinical management. Sensitivity and resistance to platinum therapy are partially determined by the tumor's intrinsic DNA repair activities, including nucleotide excision repair (NER) and base excision repair (BER). We investigated the role of the NER proteins-ERCC1, XPF, ERCC1/XPF complex-and of the BER protein DNA polymerase ß, as possible biomarkers of cisplatin (DDP) response in a platform of recently established patient-derived ovarian carcinoma xenografts (OC-PDXs). ERCC1 and DNA polymerase ß protein expressions were measured by immunohistochemistry, the ERCC1/XPF foci number was detected by proximity ligation assay (PLA) and their mRNA levels by real-time PCR. We then correlated the proteins, gene expression and ERCC1/XPF complexes with OC-PDXs' response to platinum. To the best of our knowledge, this is the first investigation of the role of the ERCC1/XPF complex, detected by PLA, in relation to the response to DDP in ovarian carcinoma. None of the proteins in the BER and NER pathways studied predicted platinum activity in this panel of OC-PDXs, nor did the ERCC1/XPF foci number. These results were partially explained by the experimental evidence that the ERCC1/XPF complex increases after DDP treatment and this possibly better associates with the cancer cells' abilities to activate the NER pathway to repair platinum-induced damage than its basal level. Our findings highlight the need for DNA functional assays to predict the response to platinum-based therapy.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

Antimetastatic and antiangiogenic activity of trabectedin in cutaneous melanoma.

Trabectedin is a marine-derived antineoplastic drug. Besides targeting the cancer cells, trabectedin has a peculiar activity on the tumor microenvironment with marked effects on the vasculature and the immune response. Because a favorable microenvironment is a key factor in the progression of cutaneous melanoma, we hypothesized that trabectedin might affect the growth and metastasis of this highly aggressive cancer. This study shows that trabectedin inhibited the subcutaneous growth of the murine melanoma B16-BL6 and K1735-M2. In line with its known activities on the environment of other tumor types, it caused a significant reduction of tumor blood vessel density and tumor-associated macrophages. Trabectedin had a significant antimetastatic activity, inhibiting the formation of lung colonies following intravenous injection of B16-BL6 or K1735-M2 cells. The drug was also active in a clinically relevant spontaneous metastasis assay, where it inhibited lung metastasis when administered before (neoadjuvant) or after (adjuvant) surgical removal of the primary tumor. Relevant to its antimetastatic activity, trabectedin inhibited melanoma cell invasiveness in vitro, associated with increased tissue inhibitor of metalloproteinase-1 production and alteration in cell shape and cytoskeleton organization. This study shows that trabectedin affects melanoma growth and metastasis, acting with tumor-dependent mechanisms on both the tumor cells and the vascular and the inflammatory tumor microenvironment.

Past-in-the-Future. Peak detection improves targeted mass spectrometry imaging.

Mass spectrometry imaging is a valuable tool for visualizing the localization of drugs in tissues, a critical issue especially in cancer pharmacology where treatment failure may depend on poor drug distribution within the tumours. Proper preprocessing procedures are mandatory to obtain quantitative data of drug distribution in tumours, even at low intensity, through reliable ion peak identification and integration. We propose a simple preprocessing and quantification pipeline. This pipeline was designed starting from classical peak integration methods, developed when "microcomputers" became available for chromatography, now applied to MSI. This pre-processing approach is based on a novel method using the fixed mass difference between the analyte and its 5 d derivatives to set up a mass range gate. We demonstrate the use of this pipeline for the evaluating the distribution of the anticancer drug paclitaxel in tumour sections. The procedure takes advantage of a simple peak analysis and allows to quantify the drug concentration in each pixel with a limit of detection below 0.1 pmol mm-2 or 10 µg g-1. Quantitative images of paclitaxel distribution in different tumour models were obtained and average paclitaxel concentrations were compared with HPLC measures in the same specimens, showing <20% difference. The scripts are developed in Python and available through GitHub, at github.com/FrancescaFalcetta/Imaging_of_drugs_distribution_and_quantifications.git.