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INTRODUCTION Despite significant improvements in advanced melanoma therapy, there is still a pressing need for innovative therapies. Here, we optimized a method for testing the drug sensitivity of PDOCs and evaluated whether this preclinical model could be a valid tool for rapidly determining the patient’s tumor response profile to approved and alternative therapies. METHODS PDOCs were generated from melanoma metastases (> 60 specimens) after mechanical or enzymatic dissociation. Tissue fragments ( RESULTS The first experiments were set up to validate PDOCs as predictive preclinical models of patient response to therapy. We tested approved melanoma therapies (i.e., the combination of BRAF and MEK inhibitors, or anti-PD-1 antibodies) on the same melanoma specimens at the IDI and IEO institutes. More than 10 different samples were compared, finding reproducible results between the two Institutions. Furthermore, drug response in PDOCs was comparable to the clinical response of matched patients undergoing the tested therapy, demonstrating PDOCs are reliable predictive tools. Then, we used primary cell lines to screen a pool of ten different targeted agents selected through functional screening of a bioactive library of 512 compounds on patient-derived xenografts, and based on known drug resistance mechanisms in melanoma. The three best-performing compounds were subsequently studied in PDOCs as single agents or in combination with immunotherapy. We found high heterogeneity of drug efficacy in different melanoma samples with no obvious correlation to BRAF or NRAS mutations, metastasis type, or patients’ prior therapies. Of note, in some cases, the combination with an anti-PD-1 inhibitor significantly improved the efficacy of one or more of the three drugs. CONCLUSIONS We optimized and validated PDOCs as personalized preclinical models suitable for assessing the drug sensitivity/resistance profile of individual patient-derived melanomas. By retaining tumor stromal components and heterogeneity, PDOCs could be used to predict patients’ clinical response to currently approved agents, helping oncologists expedite decision-making when several treatment options are possible. Furthermore, the model could be used to evaluate the effectiveness of alternative treatments on tumors resistant to approved therapies, and to explore new drug combinations. Citation Format: Antonella Bresin, Fiorenza Lotti, Lauretta Levati, Pier F. Ferrucci, Francesca Ricci, Francesco Scicchitano, Zorika C. Di Rocco, Giandomenico Russo, Luisa Lanfrancone. Patient-derived organotypic cultures (PDOCs) from melanoma metastases as a precision medicine test to improve patient management. [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 5500.

Efficacy, safety, together with molecular and immunological biomarkers were studied in a sequential clinical trial of neoadjuvant immunotherapy, surgery and adjuvant immunotherapy in locally advanced or oligometastatic melanoma patients (pts), within an open label, single arm and two sites study. Treatment schedule consisted in four primary cycles of inverted dose ipilimumab 1 mg/kg and nivolumab 3 mg/kg every 3 weeks, followed by radical surgery and adjuvant nivolumab 480 mg every 4 weeks for 6 cycles. Primary objective was pathological complete remission (pCR) rate, according to International Neoadjuvant Melanoma Consortium (INMC) criteria, while secondary objectives were: safety, feasibility and efficacy; QoL; identification of biomarkers of response and resistance; degree of immune activation; longitudinal evaluation of the gut microbiome. From March 2019 to April 2021, 43 pts were enrolled in the trial and, with an intent to treat of 35 pts, 34 completed the primary phase, 31 received surgery and 28 completed the adjuvant phase. Four pts were withdrawn during primary phase for progression (2), toxicity (1) and consent withdrawal (1). Study primary endpoint has been met since 20/31 pts undergoing surgery reached a pCR/near pCR (65%), 4/31 (13%) a pathological partial remission (pPR) and 7/31 (22%) pts a pathological no response. With a median follow-up of 17 months, 33/35 pts are alive. Treatment failure occurred in 9 pts: 2 pts progressed during primary phase and did not undergo surgery; 7 pts progressed during adjuvant (3 pts) or follow-up phase (4 pts). Six out of these 7 pts were classified as pNR at surgery, while the other, classified as pCR, did not receive adjuvant therapy. Both pts in stage IV relapsed. Unfortunately, one pt died for ischemic stroke after 5 months from adjuvant therapy while on CR. Treatment related toxicities were mainly G1-2 and only 6 pts (17%) developed G3-4 adverse events (AE): 3 transaminitis, 1 pneumonitis, 1 myocarditis, 1 CPK increase and 1 dermatomyositis. Translational studies on samples collected before, during therapy and at progression have been performed: whole exome sequencing and gut microbiota dynamics on longitudinal samples showed some relationships with responses and developing resistances. These data, never presented elsewhere previously, are in part new and in part confirmatory of immunological or molecular signatures described by other groups. In conclusion, primary immunotherapy with Ipilimumab/Nivolumab in pts affected by locally advanced/oligometastatic melanoma is able to achieve an elevated pCR/near pCR rate which appears to be predictive of long term relapse free survival. Translational data analyzed longitudinally on each patient can allow for a better selection of pts, giving new insight on the mechanisms of melanoma progression and resistance. Citation Format: Pier Francesco Ferrucci, Bruno Achutti Duso, Luigi Nezi, Luca Mazzarella, Fiorenza Lotti, Sara Gandini, Gianmarco Orsolini, Elisabetta Pennacchioli, Patrizia Gnagnarella, Teresa Manzo, Simone Ribero, Maria Teresa Fierro, Rebecca Senetta, Concetta Riviello, Virginia Caliendo, Pietro Quaglino, Massino Barberis, Giuseppina Bonizzi, Emilia Cocorocchio. Neoadjuvant ipilimumab/nivolumab in locally advanced or oligometastatic melanoma: An open label, single arm, multi-institutional clinical study with molecular and immunological biomarker’s analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT167.

Metastases are the primary cause of cancer-related deaths. The underlying molecular and biological mechanisms remain, however, elusive, thus preventing the design of specific therapies. In melanomas, the metastatic process is influenced by the acquisition of metastasis-associated mutational and epigenetic traits and the activation of metastatic-specific signaling pathways in the primary melanoma. In the current study, we investigated the role of an adaptor protein of the Shc family (ShcD) in the acquisition of metastatic properties by melanoma cells, exploiting our cohort of patient-derived xenografts (PDXs). We provide evidence that the depletion of ShcD expression increases a spread cell shape and the capability of melanoma cells to attach to the extracellular matrix while its overexpression switches their morphology from elongated to rounded on 3D matrices, enhances cells&rsquo, invasive phenotype, as observed on collagen gel, and favors metastasis formation in vivo. ShcD overexpression sustains amoeboid movement in melanoma cells, by suppressing the Rac1 signaling pathway through the confinement of DOCK4 in the cytoplasm. Inactivation of the ShcD signaling pathway makes melanoma cells more sensitive to therapeutic treatments. Consistently, ShcD expression predicts poor outcome in a cohort of 183 primary melanoma patients.

Treatment of metastatic melanoma has been recently revolutionized by the use of targeted and immune inhibitors, though efficiently working only in a fraction of patients. Adverse effects still represent major challenges, as well as the rapid development of drug resistance upon treatment, thus suggesting that it is of utmost importance to elucidate additional molecular pathways and targets to better understand the biology of this tumor. In vivo RNAi screens done in patient-derived xenografts (PDXs) that fully phenocopy the original tumor of the patient represent a powerful tool to identify new putative essential and druggable candidates, and to repurpose drugs already approved for other indications. To this end we have recently generated a platform of melanoma PDXs with which we can recapitulate both melanoma growth and metastasization. We have performed in vivo shRNA screens, where we have found numerous and patient-specific epigenetic genes essential for tumor growth in vivo. With this platform, we have isolated novel frailties whose depletion favors cell senescence. Preliminary data using shRNA libraries of actionable genes for which drugs are already available suggest that specific drugs can be repurposed for melanoma treatment. We have proved that this approach can work in vivo, and we propose to convert it in an in vitro assay to rapidly translate new discoveries to the patient. Note: This abstract was not presented at the conference. Citation Format: Federica Marocchi, Letizia Granieri, Fiorenza Lotti, Fernando Palluzzi, Jole Costanza, Laura Riva, Daniela Bossi, Luisa Lanfrancone. New treatment opportunities by in vivo and in vitro screening approaches in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A15.

In search for individualized predictors of stroke recovery, the Val66Met polymorphism of the brain-derived neurotrophic factor (BDNF) is attracting great interest, because it has a negative impact on neurotrophin function. Since stroke recovery relies on brain plastic processes, on which BDNF is permissive, the dominant thought is in favor of a worse recovery in Met carriers. Conversely, we suggest that Met carriers do not differ in terms of absolute ability to recover from stroke, but they do differ on the way they recover. In particular, Met carriers rely more on subcortical plasticity, while ValVal patients more on intracortical plastic processes. Indeed, the direct evidence of impaired Met carrier recovery is inconsistent, as a high worldwide diffusion of the polymorphism suggests. The plasticity taking place in cortex, which is the one targeted by noninvasive brain stimulation strategies aimed at enhancing recovery, is less pronounced in Met carrier stroke patients, who have instead spared global recovery potential. Enhanced subcortical plasticity sustains better stroke recovery of Met carrier mice: this may also happen in humans, explaining the weaker interhemispheric cortical excitability imbalance recently described in Met carriers. Thus, BDNF haplotype determines mechanisms and structures involved in stroke recovery. The less pronounced cortical plasticity of Met carrier implies that plastic changes induced by interventional neurophysiological protocols would be better predictors of ValVal chronic outcome and those protocols would be more effective to boost their recovery. Other strategies, more focused on subcortical mechanisms, should be used in Met carriers.

Colorectal cancer (CRC) remains a leading killer in the U.S. with resistance to treatment as the largest hurdle to cure. Colorectal cancer-initiating cells (CICs) are a self-renewing tumor population that contribute to tumor relapse. Here, we report that patient-derived CICs display relative chemoresistance compared with differentiated progeny. In contrast, conventional cell lines failed model therapeutic resistance. CICs preferentially repaired chemotherapy-induced DNA breaks, prompting us to interrogate DNA damage pathways against which pharmacologic inhibitors have been developed. We found that CICs critically depended on the key single-strand break repair mediator, poly(ADP-ribose) polymerase (PARP), to survive treatment with standard-of-care chemotherapy. Small molecule PARP inhibitors (PARPi) sensitized CICs to chemotherapy and reduced chemotherapy-treated CIC viability, self-renewal, and DNA damage repair. Although PARPi monotherapy failed to kill CICs, combined PARPi therapy with chemotherapy attenuated tumor growth in vivo. Clinical significance of PARPi for CRC patients was supported by elevated PARP levels in colorectal tumors compared with normal colon, with further increases in metastases. Collectively, our results suggest that PARP inhibition serves as a point of fragility for CICs by augmenting therapeutic efficacy of chemotherapy. Stem Cells 2019;37:42–53

The acute phase of stroke is accompanied by functional changes in the activity and interplay of both hemispheres. In healthy subjects, gender is known to impact the functional brain organization.We investigated whether gender influences also acute stroke functional changes. In thirty-five ischemic stroke patients, we evaluated the excitability of the affected (AH) and unaffected hemisphere (UH) by measuring resting and active motor threshold and motor-evoked potential amplitude under baseline conditions and after intermittent theta burst stimulation (iTBS) of AH. We also computed an index of the excitability balance between the hemispheres, laterality indexes (LI), to evidence hemispheric asymmetry. Active motor threshold differed significantly between AH and UH only in the male group (p=0.004), not in females (p>0.200), and both LIAMT and LIRMT were significantly higher in males than in females (respectively p=0.033 and p=0.042). LTP-like activity induced by iTBS in AH was more frequent in females. Gender influences the functional excitability changes that take place after human stroke and the level of LTP that can be induced by repetitive stimulation. This knowledge is of high value in the attempt of individualizing to different genders any non-invasive stimulation strategy designed to foster stroke recovery.

We investigated the membrane localization of CD95 in type I and type II cells, which differ in their ability to recruit and activate caspase-8. We found that CD95 was preferentially located in lipid rafts of type I cells, while it was present both in raft and non-raft plasma membrane sub-domains of type II cells. After stimulation, CD95 located in phospholipid-rich plasma membrane was recruited to lipid rafts in both types of cells. Similarly, CD95 cross-linking resulted in caspase-independent translocation of FADD/MORT1 and caspase-8 to the lipid rafts, which was prevented by a death domain-defective receptor. CD95 internalization was then rapid in type I and delayed in type II cells and showed a substantial correlation with the kinetics of Fas-associated death domain (FADD)and caspase-8 recruitment to lipid rafts. Finally, electron microscopy analysis showed that after CD95 stimulation lipid rafts aggregated in large clusters that were internalized in endosomal vesicles, where caspase-8 underwent massive processing. Taken together, our data demonstrate that CD95 death-inducing signaling complex formation and internalization in type I and type II cells occur in lipid rafts, which are a major site of caspase-8 activation.

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133(+) cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 10(4) lung cancer CD133(+) cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133(+) cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133(+) cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments. (Cancer Res 2005; 65(24): 11469-77)