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Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) can be considered as a spectrum of the same disease entity, representing one of the most common adult soft tissue sarcoma (STS) of the extremities. While MFS is rarely metastasizing, it shows an extremely high rate of multiple frequent local recurrences (50–60% of cases). On the other hand, UPS is an aggressive sarcoma prone to distant recurrence, which is correlated to a poor prognosis. Differential diagnosis is challenging due to their heterogeneous morphology, with UPS remaining a diagnosis of exclusion for sarcomas with unknown differentiation lineage. Moreover, both lesions suffer from the unavailability of diagnostic and prognostic biomarkers. In this context, a genomic approach combined with pharmacological profiling could allow the identification of new predictive biomarkers that may be exploited for differential diagnosis, prognosis and targeted therapy, with the aim to improve the management of STS patients. RNA-Seq analysis identified the up-regulation of MMP13 and WNT7B in UPS and the up-regulation of AKR1C2, AKR1C3, BMP7, and SGCG in MFS, which were confirmed by in silico analyses. Moreover, we identified the down-regulation of immunoglobulin genes in patient-derived primary cultures that responded to anthracycline treatment compared to non-responder cultures. Globally, the obtained data corroborated the clinical observation of UPS as an histotype refractory to chemotherapy and the key role of the immune system in determining chemosensitivity of these lesions. Moreover, our results confirmed the validity of genomic approaches for the identification of predictive biomarkers in poorly characterized neoplasms as well as the robustness of our patient-derived primary culture models in recapitulating the chemosensitivity features of STS. Taken as a whole, this body of evidence may pave the way toward an improvement of the prognosis of these rare diseases through a treatment modulation driven by a biomarker-based patient stratification.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Neuroendocrine tumors (NETs) are rare neoplasms frequently characterized by an upregulation of the mammalian rapamycin targeting (mTOR) pathway resulting in uncontrolled cell proliferation. The mTOR pathway is also involved in skeletal muscle protein synthesis and in adipose tissue metabolism. Everolimus inhibits the mTOR pathway, resulting in blockade of cell growth and tumor progression. The aim of this study is to investigate the role of body composition indexes in patients with metastatic NETs treated with everolimus. The study population included 30 patients with well-differentiated (G1-G2), metastatic NETs treated with everolimus at the IRCCS Romagnolo Institute for the Study of Tumors (IRST) “Dino Amadori”, Meldola (FC), Italy. The body composition indexes (skeletal muscle index [SMI] and adipose tissue indexes) were assessed by measuring on a computed tomography (CT) scan the cross-sectional area at L3 at baseline and at the first radiological assessment after the start of treatment. The body mass index (BMI) was assessed at baseline. The median progression-free survival (PFS) was 8.9 months (95% confidence interval [CI]: 3.4–13.7 months). The PFS stratified by tertiles was 3.2 months (95% CI: 0.9–10.1 months) in patients with low SMI (tertile 1), 14.2 months (95% CI: 2.3 months-not estimable [NE]) in patients with intermediate SMI (tertile 2), and 9.1 months (95% CI: 2.7 months-NE) in patients with high SMI (tertile 3) (p = 0.039). Similarly, the other body composition indexes also showed a statistically significant difference in the three groups on the basis of tertiles. The median PFS was 3.2 months (95% CI: 0.9–6.7 months) in underweight patients (BMI ≤ 18.49 kg/m2) and 10.1 months (95% CI: 3.7–28.4 months) in normal-weight patients (p = 0.011). There were no significant differences in terms of overall survival. The study showed a correlation between PFS and the body composition indexes in patients with NETs treated with everolimus, underlining the role of adipose and muscle tissue in these patients.

Head and neck cancers (HNCs) represent the sixth most widespread malignancy worldwide. Surgery, radiotherapy, chemotherapeutic and immunotherapeutic drugs represent the main clinical approaches for HNC patients. Moreover, HNCs are characterised by an elevated mutational load; however, specific genetic mutations or biomarkers have not yet been found. In this scenario, personalised medicine is showing its efficacy. To study the reliability and the effects of personalised treatments, preclinical research can take advantage of next-generation sequencing and innovative technologies that have been developed to obtain genomic and multi-omic profiles to drive personalised treatments. The crosstalk between malignant and healthy components, as well as interactions with extracellular matrices, are important features which are responsible for treatment failure. Preclinical research has constantly implemented in vitro and in vivo models to mimic the natural tumour microenvironment. Among them, 3D systems have been developed to reproduce the tumour mass architecture, such as biomimetic scaffolds and organoids. In addition, in vivo models have been changed over the last decades to overcome problems such as animal management complexity and time-consuming experiments. In this review, we will explore the new approaches aimed to improve preclinical tools to study and apply precision medicine as a therapeutic option for patients affected by HNCs.

Background: Neuroendocrine neoplasms (NENs) are a subgroup of poorly characterized rare tumors with challenging management. Identification of clinically useful biomarkers for patient stratification and treatment tailoring is urgently needed. This study proposes the use of near-patient models based on autologous extracellular matrix (aECM)-scaffolds and zebrafish (ZF) xenografts integrated with next generation multi-omics analysis for the phenotype and genotype mapping of NEN cells. The aim is to uncover molecular signatures associated with disease aggressiveness and to detect oncogenic drivers for targeted treatment. Methods: Primary tumor cells isolated from surgical specimens of NEN patients were cultured in aECM scaffolds or injected into ZF embryos. Autologous ECM-scaffolds were synthesized starting from the patients’ matrices and recellularized with primary cells derived from matched patients. In aECM scaffolds we evaluated tumor cell proliferation and neuroendocrine differentiation. In ZF we evaluated cell angiogenic ability and distant invasiveness. For each primary culture, we performed exome and transcriptome analysis of the original tumor tissue compared to the matched healthy counterpart. Results: We demonstrated that ZF xenografting faithfully recreates the behavior that tumor cells exhibit in patients. Specifically, we derived a primary culture from an ileal grade 1 neuroendocrine tumor from both the primary and the metastatic lesions. When injected into ZF embryos, cells derived from the primary tumor displayed low invasive ability with a metastatic rate of only 23%. Conversely, cells derived from the metastatic lesion were able to invade the ZF circulation and form distant metastases in 79% of embryos. Consecutively, we derived three primary cultures of neuroendocrine Merkel cell carcinoma (MCC). The 3 MCC cultures into ZF embryos showed high aggressiveness with metastatic rates of 81%, 50% and 64%. For one culture, angiogenic ability was also observed. RNA-sequencing of these aggressive MCC specimens highlighted the involvement of the following pathways: ECM remodeling, TNF-ⲁ and PI3K-Akt signaling, epithelial-mesenchymal transition and regulation of apoptotic processes. Next, we demonstrated that aECM-scaffolds morphologically resemble a native tumor tissue, and tumor cells, once seeded on them, preserved the expression of neuroendocrine markers. Conclusions: Here we developed innovative neuroendocrine tumor models that are expected to generate new knowledge on the disease molecular determinants. All identified genomic and genetic alterations will be correlated with the clinical outcomes of enrolled patients, to validate their clinical significance and to ultimately introduce a biomarker-based approach for NEN patients. Citation Format: Chiara Calabrese, Giacomo Miserocchi, Silvia Vanni, Alessandro De Vita, Alberto Bongiovanni, Chiara Spadazzi, Claudia Cocchi, Nicoletta Ranallo, Federica Pieri, Michela Tebaldi, Flavia Foca, Giorgio Ercolani, Davide Cavaliere, Carlo Alberto Pacilio, Giovanni Martinelli, Laura Mercatali, Chiara Liverani. Autologous ECM-composed scaffolds and zebrafish PDXs for individualized theranostics in rare neuroendocrine neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3076.

Background: Liposarcoma (LPS) encompass one of most common soft tissue sarcoma (STS) histological subtypes accounting for 15% of all cases. They can be divided into four entities including atypical lipomatous tumor or well-differentiated LPS (ALT/WDLPS), dedifferentiated LPS (DDLPS), myxoid LPS (MLPS) and pleomorphic LPS (PLS). Their heterogeneity is reflected in their morphology, molecular landscape, prognosis and clinical behavior. Current treatment options for localized disease include surgery, (neo)adjuvant radiotherapy and chemotherapy; for the metastatic setting chemotherapy represents the cornerstone but its role needs to be elucidated. The unavailability of predictive biomarkers makes the management of these mesenchymal diseases even more challenging. Recent evidences have shed light on the key role of cyclin-dependent kinase 4 (CDK4) in cancer cells proliferation. Methods: This study involved 21 adult patients affected by liposarcoma (n= 5 ALT/WDLPS and n= 16 DDLPS). IHC analyses of CDK4 and MDM2 were performed on FFPE surgically resected specimens by experienced sarcoma pathologists. Moreover, a patient-derived DDLPS cell line was established and pharmacological profiling was performed. Next, standard and innovative drugs currently used for LPS management were assessed in both 2D and 3D culture systems. Finally, in silico analyses based on public repositories on STS were carried out including 260 sarcoma patients. Results: IHC results highlighted an higher expression of CDK4 and MDM2 biomarkers in DDLPS compared to ALT/WDLSP. Moreover, although CDK4 and MDM2 are codified by the same genomic region, their expression did not correlate (e.g. 100% of CDK4 and 2% of MDM2 in the same patient). Therefore we hypothesized that even exhibiting a low MDM2 expression, a patient could benefit from CDK4 inhibition. Furthermore, our data showed that in DDLPS cases CDK4 expression ranged from 90% to 100% in spermatic cord, skin and abdomen while it ranged from 5% to 50% in the retroperitoneum. The above observations prompted us to hypothesize a correlation of CDK4 expression with specific anatomical sites. Moreover, pharmacological profiling showed a synergistic effect exerted by sequential treatment with palbociclib and some chemotherapeutics including trabectedin, dacarbazine, eribulin and lenvatinib. Indeed we observed a 10% increase in cell proliferation inhibition compared to standard treatment. In addition, in silico analyses revealed the role of CDK4 as negative prognostic biomarker for PFS and OS in STS. Conclusions: Our study highlighted the promising role of CDK4 in the management of LPS patients. In particular, we pointed out encouraging results of sequential treatment combining CDK4 inhibitor palbociclib and chemotherapy. Furthermore, preliminary analyses highlighted the involvement of this biomarker as a potential prognostic tool for STS. Citation Format: Silvia Vanni, Graziana Gallo, Valentina Fausti, Giacomo Miserocchi, Chiara Liverani, Chiara Spadazzi, Claudia Cocchi, Chiara Calabrese, Giovanni De Luca, Massimo Bassi, Manlio Gessaroli, Angelo Campobassi, Federica Pieri, Giorgio Ercolani, Davide Cavaliere, Lorena Gurrieri, Nada Riva, Giovanni Martinelli, Laura Mercatali, Alessandro De Vita. Dissecting the role of CDK4 in liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5993.

Sarcomas are heterogeneous and rare malignant tumors that develop from connective tissues. Myxofibrosarcoma (MFS) and Osteosarcoma (OS) are the main representative subtypes of Soft tissues and Bone sarcomas. Conventional treatment consists of surgical resection and neo/adjuvant chemotherapy. However, a challenging clinical feature is a high local recurrence rate, calling for the development of novel strategies to eradicate MFS and OS. Fluorescence-guided surgery (FGS), coupled with photodynamic therapy (PDT), enables to detect and eradicate malignant tissues using a light-sensitive photosensitizer (PS). These compounds could be retained by neoplastic cells to a greater extent than by healthy tissue. Thus, when photoactivated, they lead to cancer cell death, minimizing normal tissues toxicity. The project aims to provide a preclinical proof of concept of FGS and PDT for MFS and OS, allowing us to pave the way for a more effective treatment strategy. A multimodel preclinical platform of MFS and OS comprising 2D, 3D-collagen based scaffold was developed. A MFS stabilized cell line (IM-MFS-1) and four OS cell lines (Saos-2, U-2 OS, MG-63, 143B) were used. NIH-3T3 fibroblast cell line was used as healthy counterpart. We tested a panel of synthetic PSs: Toluidine Blue, 5-Aminolevulinic Acid hydrochloride (ALA), Methylene Blue, Chlorin e6 (Ce6), Pheophorbide a (Pba) and Al(III) Phthalocyanine Chloride Tetrasulfonic Acid (AlPcS4) that can be photoactivated with light in the 630-660 nm wavelengths’ range. The PSs were ranked on (a) selective uptake by tumor cells, respect to healthy cells and (b) the cytotoxic effect, after photoactivation. PSs cell uptake was evaluated by Countess II FL automated cell counter. Cytotoxicity was evaluated by WST-1 and Presto Blue assays. For each PS we determined the highest concentration, causing less than 50% dark toxicity in IM-MFS-1, OS cell lines and fibroblast NIH-3T3, seeded in 2D and 3D collagen-based models. Then, we evaluated the cell viability upon light irradiation (λ=630 or 660 nm; 10 mW/cm^2, 5 min). For all PSs, dark toxicity was very low. However, only Ce6 was effectively uptaken by tumor cells (% uptake > 60%) and not by NIH-3T3 cells (% uptake = 27%). Moreover, upon photoactivation, Ce6 induced significantly higher cell death in cancer cells as compared to NIH-3T3 cells, e.g. 1-3% cell viability in tumor cells and 76% cell viability in NIH-3T3. Results from our multimodel platform highlight PDT as a powerful strategy for the treatment of MFS and OS. Among the screened PSs, Ce6 is the most promising, showing a remarkable selectivity in tumor cells uptake and cytotoxicity. To increase the clinical significance of our approach, we will confirm its efficacy in patient-derived primary cultures; xenograft zebrafish in vivo embryos will be used to evaluate how PDT affects the disease aggressiveness and metastatic potential. Citation Format: Chiara Spadazzi, Micaela Pannella, Chiara Bellotti, Elisa Martella, Silvia Vanni, Alessandro De Vita, Chiara Liverani, Giacomo Miserocchi, Claudia Cocchi, Chiara Calabrese, Valentina Fausti, Roberto Casadei, Federica Pieri, Ania Naila Guerrieri, Giovanni Martinelli, Greta Varchi, Enrico Lucarelli, Laura Mercatali, Toni Ibrahim. A multimodel preclinical platform to evaluate photodynamic therapy as a strategy to reduce local recurrence in myxofibrosarcoma and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2419.

Adult rhabdomyosarcoma (RMS) represents an uncommon entity with an incidence of less than 3% of all soft tissue sarcomas (STS). Consequently, the natural history and the clinical management of this disease are infrequently reported. In order to fill this gap, we investigated the molecular biology of an adult RMS case series. The expression of epithelial mesenchymal transition-related gene and chemoresistance-related gene panels were evaluated. Moreover, taking advantage of our STS translational model combining patient-derived primary culture and 3D-scaffold, the pharmacological profile of an adult head and neck sclerosing RMS was assessed. Furthermore, NGS, microsatellite instability, and in silico analyses were carried out. RT-PCR identified the upregulation of CDH1, SLUG, MMP9, RAB22a, S100P, and LAPTM4b, representing promising biomarkers for this disease. Pharmacological profiling showed the highest sensitivity with anthracycline-based regimen in both 2D and 3D culture systems. NGS analysis detected RAB3IP-HMGA2 in frame gene rearrangement and FGFR4 mutation, microsatellite instability analysis did not detect any alteration. In silico analysis confirmed the mutation of FGFR4 as a promising marker for poor prognosis and a potential therapeutic target. We report for the first time the molecular and pharmacological characterization of rare entities of adult head and neck and posterior trunk RMS. These preliminary data could shed light on this poorly understood disease.

How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial–mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal–epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal–epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal–epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.

Myxofibrosarcoma (MFS) is a common entity of adult soft tissue sarcomas (STS) characterized by a predilection of the extremities and a high local recurrence rate. Originally classified as a myxoid variant of malignant fibrous histiocytoma, this musculoskeletal tumor has been recognized since 2002 as a distinct histotype showing a spectrum of malignant fibroblastic lesions with myxoid stroma, pleomorphism and curvilinear vessels. Currently, the molecular pathogenesis of MFS is still poorly understood and its genomic profile exhibits a complex karyotype with a number of aberrations including amplifications, deletions and loss of function. The diagnosis is challenging due to the unavailability of specific immunohistochemical markers and is based on the analysis of cytomorphologic features. The mainstay of treatment for localized disease is represented by surgical resection, with (neo)-adjuvant radio- and chemotherapy. In the metastatic setting, chemotherapy represents the backbone of treatments, however its role is still controversial and the outcome is very poor. Recent advent of genomic profiling, targeted therapies and larger enrollment of patients in translational and clinical studies, have improved the understanding of biological behavior and clinical outcome of such a disease. This review will provide an overview of current diagnostic pitfalls and clinical management of MFS. Finally, a look at future directions will be discussed.

To improve the success rate of current preclinical drug trials, there is a growing need for more complex and relevant models that can help predict clinical resistance to anticancer agents. Here, we present a three‐dimensional (3D) technology, based on biomimetic collagen scaffolds, that enables the modeling of the tumor hypoxic state and the prediction of in vivo chemotherapy responses in terms of efficacy, molecular alterations, and emergence of resistance mechanisms. The human breast cancer cell lines MDA‐MB‐231 (triple negative) and MCF‐7 (luminal A) were treated with scaling doses of doxorubicin in monolayer cultures, 3D collagen scaffolds, or orthotopically transplanted murine models. Lineage‐specific resistance mechanisms were revealed by the 3D tumor model. Reduced drug uptake, increased drug efflux, and drug lysosomal confinement were observed in triple‐negative MDA‐MB‐231 cells. In luminal A MCF‐7 cells, the selection of a drug‐resistant subline from parental cells with deregulation of p53 pathways occurred. These cells were demonstrated to be insensitive to DNA damage. Transcriptome analysis was carried out to identify differentially expressed genes (DEGs) in treated cells. DEG evaluation in breast cancer patients demonstrated their potential role as predictive biomarkers. High expression of the transporter associated with antigen processing 1 (TAP1) and the tumor protein p53‐inducible protein 3 (TP53I3) was associated with shorter relapse in patients affected by ER+ breast tumor. Likewise, the same clinical outcome was associated with high expression of the lysosomal‐associated membrane protein 1 LAMP1 in triple‐negative breast cancer. Hypoxia inhibition by resveratrol treatment was found to partially re‐sensitize cells to doxorubicin treatment. Our model might improve preclinical in vitro analysis for the translation of anticancer compounds as it provides: (a) more accurate data on drug efficacy and (b) enhanced understanding of resistance mechanisms and molecular drivers.

Bone is one of the most preferred sites of metastatic spread from different cancer types, including breast cancer. However, different breast cancer subtypes exhibit distinct metastatic behavior in terms of kinetics and anatomic sites of relapse. Despite advances in the diagnosis, the identification of patients at high-risk of bone recurrence is still an unmet clinical need. We conducted a retrospective analysis, by gene expression and immunohistochemical assays, on 90 surgically resected breast cancer samples collected from patients who experienced no evidence of distant metastasis, bone or visceral metastasis in order to identify a primary tumor-derived marker of bone recurrence. We identified trefoil factor-1 (pS2 or TFF1) as strictly correlated to bone metastasis from ER+ breast cancer. In silico analysis was carried out to confirm this observation, linking gene expression data with clinical characteristics available from public clinical datasets. Then, we investigated TFF1 function in ER+ breast cancer tumorigenesis and bone metastasis through xenograft in vivo models of MCF 7 breast cancer with gain and loss of function of TFF1. As a response to microenvironmental features in primary tumors, TFF1 expression could modulate ER+ breast cancer growth, leading to a less proliferative phenotype. Our results showed it may not play a role in late stages of bone metastasis, however further studies are warranted to understand whether it could contribute in the early-stages of the metastatic cascade. In conclusion, TFF1 upregulation in primary ER+ breast cancer could be useful to identify patients at high-risk of bone metastasis. This could help clinicians in the identification of patients who likely can develop bone metastasis and who could benefit from personalized treatments and follow-up strategies to prevent metastatic disease.

Conventional 2D cell culture, a traditional tool in pre-clinical studies, can hardly be regarded as a representation of a natural cell microenvironment. In this respect, it might result in altered cellular behaviors. To overcome such a limitation, different approaches have been tested to conduct more representative in vitro studies. In particular, the use of 3D cell culture introduces variables, such as cell-cell and cell-extracellular matrix interactions; cell features such as survival, proliferation and migration are consequently influenced. For an example, an enhanced drug resistance and increased invasiveness are shown by cancer cells when cultured in 3D versus 2D conventional culture models. In this setting however, non-uniform cell distribution and biological behaviors appear throughout the scaffold, due to reduced diffusion of oxygen and nutrients. Perfusion in bioreactor systems can be used to improve medium transport. In this line of reasoning, this study proposes a breast cancer cell culture model sustained by an integrated approach that couples a 3D environment and a fluid perfusion. This model improves viability and uniformness of cell distribution, while inducing morphological, functional and molecular cancer cell remodeling.

Background Soft tissue sarcomas (STS) are a rare group of solid neoplasm including among others liposarcoma, leiomyosarcoma (L-sarcoma) and undifferentiated pleomorphic sarcoma (UPS) entities. The current first-line treatment is represented by anthracycline based- regimens, second-line may include trabectedin. Currently the activity of trabectedin and its mechanism of action is not completely elucidated. Methods Taking the advantages of our 3D patient-derived primary culture translational model we performed genomic-, chemobiogram, proteomic- and in vivo analysis in a UPS culture (S1). Furthermore pharmacological profiling of a UPS and L-sarcoma patient-derived case series and in silico analysis were carried out. Results Trabectedin exhibited an increased activity in 3D respect to 2D cultures suggesting an extracellular matrix (ECM) and timp1 involvement in its mechanism of action. Moreover 3D S1 xenotranspanted zebrafish model showed an increased sensitivity to trabectedin. Finally the results were further validated in a UPS and L-sarcoma case series. Conclusions Taken together these results confirmed the activity of trabectedin in these STS histotypes. Moreover the data underline the ECM involvement in the cytotoxic effect mediated by trabectedin and could open the door for researches aimed to focus on the patient setting that could benefit from this agent.

In the field of oncology research, a deeper understanding of tumor biology has shed light on the role of environmental conditions surrounding cancer cells. In this regard, targeting the tumor microenvironment has recently emerged as a new way to access this disease. In this work, a novel extracellular matrix (ECM)-targeting nanotherapeutic was engineered using a lipid-based nanoparticle chemically linked to an inhibitor of the ECM-related enzyme, lysyl oxidase 1 (LOX), that inhibits the crosslinking of elastin and collagen fibers. We demonstrated that, when the conjugated vesicles were loaded with the chemotherapeutic epirubicin, superior inhibition of triple negative breast cancer (TNBC) cell growth was observed both in vitro and in vivo. Moreover, in vivo results displayed prolonged survival, minimal cytotoxicity, and enhanced biocompatibility compared to free epirubicin and epirubicin-loaded nanoparticles. This all-in-one nano-based ECM-targeting chemotherapeutic may provide a key-enabling technology for the treatment of TNBC.

Background: Neuroendocrine neoplasias (NENs) are a rare group of tumors with different prognosis and response to therapy. Their heterogeneity is dependent on the site of origin, morphology, and Ki67. Temozolomide (TEM) appears to be active in metastatic NENs (mNENs) but there is limited evidence about its efficacy in gastrointestinal NENs. We analyzed “real-world” data on the use of TEM alone or in association with capecitabine (CAPTEM) in patients with mNENs. Patients and Methods: One hundred consecutive patients with advanced NENs treated with TEM or CAPTEM between 2009 and 2019 were included. A pretreatment tumor growth rate (TGR0) was calculated. Overall survival (OS), progression-free survival (PFS), tolerance, objective response rate (ORR), and disease control rate (DCR) were analyzed. A propensity score analysis and inverse probability of treatment weights for Cox regression models were used. Results: TEM-based therapy was administered to 95 patients (26.3% CAPTEM and 83.7% TEM) with a median age of 59 years (range 26–85) years. ECOG performance status was 0–2. Carcinoid syndrome was reported in 12 (12.6%) patients. Twenty (21.1%) patients with grade (G) 3 neuroendocrine carcinoma (NEC) and 9 (9.4%) with G3 neuroendocrine tumors (NETs) were included in the analysis. Median PFS of the entire group was 10.4 months (95% confidence interval [CI]: 6.0–11.5). In multivariate analysis, a higher risk of progression was observed for NEC G3 patients (hazard ratio [HR] 2.70, 95% CI: 1.25–5.84) and for a TGR ≥19.55 (HR: 2.53, 95% CI: 1.45–4.40). Median OS was 23.4 months (95% CI: 17.0–29.0) and was similar in both treatment groups (23.9 vs. 20.5 months for TEM and CAPTEM, respectively, p = 0.585). In multivariate analysis, TGR ≥19.55 was associated with a higher risk of death (HR: 2.18, 95% CI: 1.16–4.11) than TGR Conclusions: TEM-based regimens are associated with a high DCR and a relatively tolerable toxicity profile in NENs of pancreatic, intestinal, and lung origin. Further investigation of these specific NETs is warranted in prospective clinical trials.

Background: Several phase-II trials have been designed to evaluate tyrosine kinase inhibitors (TKIs), in particular, pazopanib in neuroendocrine neoplasia (NEN), but its efficacy has not yet been demonstrated in a randomised-controlled Phase III trial. A systematic review of the published clinical trials of metastatic NEN patients could reduce the possible bias of single phase II studies. The present systematic review focuses on the efficacy and safety of pazopanib in patients with metastatic and locally advanced NEN. Methods: A systematic search in the major databases Medline/PubMed, Cochrane and Embase and in supplementary material from important international Meetings was performed to identify publications on pazopanib for the treatment of neuroendocrine neoplasia. English language was defined as a restriction. Four authors of the present review independently performed the study selection, assessed the risk of bias and extracted study data. Four published clinical trials and 2 abstracts were identified. One trial was excluded because the topic was Von-Hippel Landau disease and one abstract was eliminated because of the lack of information on meeting proceedings. Results: In all of the trials pazopanib was orally administered at a dose of 800 mg daily continuously with a 28-day cycle. The intention-to-treat population for efficacy was composed of 230 patients with a median age of 62 years. The partial response rate was 10.7% (95% confidence interval 2.6–20.5). The rate for stable disease was 79.6% (range: 61.7–92.1%) with a disease control rate (DCR) of 90.3%. Progressive disease was reported in 9.7% (range 5.2–17.6) of patients. No complete responses were observed. Median progression-free survival was 11.6 months (95% CI: 9.2–13.9). Overall survival from all the trials was 24.6 (95% CI: 18.7–40.8) months. Severe adverse events (grade III–IV) included hypertension 31%, 16% increase in AST/ALT, diarrhoea 10% and fatigue 10%. Conclusions: Pazopanib monotherapy achieved a DCR of 90.3% in patients with locally advanced and/or metastatic neuroendocrine neoplasia, with an overall response rate comparable to other TKIs and mTOR inhibitors and a safety profile similar to that of drugs of the same class.

Background:Myxofibrosarcoma (MFS), formerly considered as a myxoid variant of malignant fibrous histiocytoma, is the most common sarcoma of the extremities in adults and is characterized by a high frequency of local recurrence. The clinical behavior of MFS is unpredictable and the efficacy of chemotherapy is still not well documented. Furthermore, given the relatively recent recognition of MFS as a distinct pathologic entity its cellular and molecular biology has still not been extensively studied in patient-derived preclinical models. We examined the molecular biology and treatment outcomes of high-grade, patient-derived MFS primary cultures.Methods:A total of three patient-derived MFS primary cultures were analyzed. We evaluated the role of CD109 expression and also looked for a correlation between transforming growth factor-beta (TGF-β) expression and sensitivity of the primary cultures to different drugs.Results:CD109 was a promising marker for the identification of more aggressive high-grade MFS and a potential therapeutic target. The results also highlighted the potential role of TGF-β in chemoresistance. Pharmacological analysis confirmed the sensitivity of the cultures to chemotherapy. The most active treatments were epirubicin alone and epirubicin in combination with ifosfamide, the latter representing the current standard of care for soft tissue sarcomas (STSs), including MFS.Conclusions:Our results provide a starting point for further research aimed at improving the management of MFS patients undergoing chemotherapy.

Cancer metastasis is the leading cause of morbidity and cancer-related death. Bone is one of the most favored sites of metastatic spread, particularly from breast cancer. However, our understanding on how the host tissue can impact tumor growth is weak. This is hampered by the lack of a reliable in vitro model ables to replicate the complex microenvironment. Indeed, bone microenvironment provides physical support to cells but it can also affect cell behaviour and phenotype. We developed a hybrid 3D collagen-based scaffold functionalized with hydroxyapatite material (HA) in order to mimic natural bone features. Scaffolds were synthesized in our laboratory through collagen solution mixed and cross-linked with hydroxyapatite. Then, we cultured MDA-MB-231, a triple negative breast cancer cell line and MCF-7, an ER+ breast cancer cell line in collagen or hybrid scaffold, in order to evaluate how extracellular matrix affects cell behavior. Cell growth was evaluated by MTT cell-proliferation assay in a time course analysis. Moreover, we evaluated cell morphology and cell disposition within the scaffold by confocal microscopy. Finally, gene profiling was evaluated by qRT-PCR. MCF-7 cells acquire an organized and linear structure, whereas MDA-MB-231 an epithelial-like morphology, when cultured in hybrid scaffold. MDA-MB-231 cells proliferate faster than MCF-7 in both models. However, this is not affected by hydroxyapatite since the proliferation rate is similar when cells are cultured in collagen or hybrid scaffold. MDA-MB-231 grown in hybrid scaffold show a significant increase in RANKL/OPG ratio, pathway strictly involved in bone homeostasis. Moreover, they show a significant increased expression in JAG1, MMP2 and SNAIL1. For MCF-7, we observe a decrease in CDH1/VIM expression ratio and a significant decrease in OPG, osteoclastogenesis inhibitory factor. Overall, these results suggest a shift induced by hydroxyapatite scaffold towards an osteolytic phenotype. Whereas collagen-scaffold could better simulate in vivo primary tumor (Liverani C. et al, Sci Rep 9, 12263, 2019), we highlight that hybrid hydroxyapatite/collagen scaffold is more suitable to study breast cancer cells behaviour in bone metastasis. Then, we will implement it with a direct co-culture of osteoclast and osteoblast cells, in order to better investigate the contribution of the host microenvironment in bone metastasis. This model could provide a reliable 3D in vitro model for the study of bone metastasis mechanisms and for drug screening assays. Citation Format: Chiara Spadazzi, Laura Mercatali, Alessandro De Vita, Chiara Liverani, Claudia Cocchi, Giacomo Miserocchi, Alberto Bongiovanni, Federica Recine, Sebastiano Calpona, Nada Riva, Giandomenico Di Menna, Lorena Gurrieri, Valentina Fausti, Silvia Angela De Bonis, Giovanni Martinelli, Nicola Baldini, Toni Ibrahim. Development of a biomimetic 3D scaffold to study breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 333.

// Dino Amadori 1 , Patrizia Serra 2 , Nestory Masalu 3 , Akwilina Pangan 3 , Emanuela Scarpi 2 , Aloyce Maria Bugingo 3 , Deogratias Katabalo 3 , Toni Ibrahim 4 , Alberto Bongiovanni 4 , Giacomo Miserocchi 4 , Chiara Spadazzi 4 , Chiara Liverani 4 , Valentina Turri 5 , Rosanna Tedaldi 1 , Laura Mercatali 4 1 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Department of Oncology, Bugando Medical Center, Mwanza, Tanzania, Africa 4 Osteoncology and Rare Tumors Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 5 Healthcare Administration, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy Correspondence to: Dino Amadori, email: dino.amadori@irst.emr.it Keywords: Africans, caucasians, 25(OH)D, vitamin D pathway, vitamin D binding protein Received: September 27, 2016 Accepted: December 15, 2016 Published: January 04, 2017 ABSTRACT As total vitamin D levels are often lower in black than in white Americans, the former are frequently classified as vitamin D-deficient. To fully understand African vitamin D (25(OH)D) status, other factors should be considered, e.g. vitamin D blood carrier, vitamin D-binding protein (DBP), vitamin D receptor ( VDR ) and DBP polymorphisms. A prospective study on an indigenous black Tanzanian and a Caucasian Italian population was performed on 50 healthy donors from both populations and 35 Caucasian and 18 African breast cancer patients. 25(OH)D and DBP serum levels were analyzed by ELISA. A1012G, Cdx2 and Fok1 VDR polymorphisms and DBP polymorphisms rs4588 and rs7041 were genotyped by real-time PCR. Vitamin D and DBP levels were lower in healthy African donors than in Caucasians. Africans had a significantly higher frequency of AA and CC for Cdx2 and Fok1 polymorphisms, respectively. These allelic variants were related to a higher transcription of VDR gene and a higher activity of VDR receptor. With regard to polymorphism distribution, Africans showed innate higher levels and activity of VDR. We conclude that a strengthening of the vitamin D pathway could have a protective role against the development of breast cancer in the African population.