Publications by authors named "Paola Squarcina"

11 Publications

  • Page 1 of 1

Back to simplicity: a four-marker blood cell score to quantify prognostically relevant myeloid cells in melanoma patients.

J Immunother Cancer 2021 Feb;9(2)

Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Background: Myeloid-derived suppressor cells (MDSC), a cornerstone of cancer-related immunosuppression, influence response to therapy and disease outcomes in melanoma patients. Nevertheless, their quantification is far from being integrated into routine clinical practice mostly because of the complex and still evolving phenotypic signatures applied to define the cell subsets. Here, we used a multistep downsizing process to verify whether a core of few markers could be sufficient to capture the prognostic potential of myeloid cells in peripheral blood mononuclear cells (PBMC) of metastatic melanoma patients.

Methods: In baseline frozen PBMC from a total of 143 stage IIIc to IV melanoma patients, we first assessed the relevant or redundant expression of myeloid and MDSC-related markers by flow cytometry (screening set, n=23 patients). Subsequently, we applied the identified panel to the development set samples (n=59 patients undergoing first/second-line therapy) to obtain prognostic variables associated with overall survival (OS) and progression-free survival (PFS) by machine learning adaptive index modeling. Finally, the identified score was confirmed in a validation set (n=61) and compared with standard clinical prognostic factors to assess its additive value in patient prognostication.

Results: This selection process led to the identification of what we defined myeloid index score (MIS), which is composed by four cell subsets (CD14, CD14HLA-DR, CD14PD-L1 and CD15 cells), whose frequencies above cut-offs stratified melanoma patients according to progressively worse prognosis. Patients with a MIS=0, showing no over-threshold value of MIS subsets, had the best clinical outcome, with a median survival of >33.6 months, while in patients with MIS 1→3, OS deteriorated from 10.9 to 6.8 and 6.0 months as the MIS increased (p<0.0001, c-index=0.745). MIS clustered patients into risk groups also according to PFS (p<0.0001). The inverse correlation between MIS and survival was confirmed in the validation set, was independent of the type of therapy and was not interfered by clinical prognostic factors. MIS HR was remarkably superior to that of lactate dehydrogenase, tumor burden and neutrophil-to-lymphocyte ratio.

Conclusion: The MIS >0 identifies melanoma patients with a more aggressive disease, thus acting as a simple blood biomarker that can help tailoring therapeutic choices in real-life oncology.
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http://dx.doi.org/10.1136/jitc-2020-001167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887358PMC
February 2021

Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors.

Front Immunol 2020 13;11:262. Epub 2020 Mar 13.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity and . Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101CD56 nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56 NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101CD56 exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.
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http://dx.doi.org/10.3389/fimmu.2020.00262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082405PMC
March 2021

TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Armed Exosomes Deliver Proapoptotic Signals to Tumor Site.

Clin Cancer Res 2016 07 4;22(14):3499-512. Epub 2016 Mar 4.

Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Purpose: Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models.

Experimental Methods And Results: K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL(+) exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL(+) exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5(+) cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5(-)DR4(+)KMS11 multiple myeloma. Intratumor injection of TRAIL(+) exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL(+) exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected.

Conclusions: TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499-512. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2170DOI Listing
July 2016

Limited induction of tumor cross-reactive T cells without a measurable clinical benefit in early melanoma patients vaccinated with human leukocyte antigen class I-modified peptides.

Clin Cancer Res 2012 Dec 2;18(23):6485-96. Epub 2012 Oct 2.

Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Purpose: The progressive immune dysfunctions that occur in patients with advanced melanoma make them unlikely to efficiently respond to cancer vaccines. A multicenter randomized phase II trial was conducted to test whether immunization with modified HLA class I tumor peptides in the context of adjuvant therapy results in better immunologic responses and improved clinical outcomes in patients with early melanoma (stages IIB/C-III).

Experimental Design: Forty-three patients were enrolled to undergo vaccination (n = 22) or observation (n = 21). The vaccine included four HLA-A*0201-restricted modified peptides (Melan-A/MART-1([27L]), gp100([210M]), NY-ESO-1([165V]), and Survivin([97M])) emulsified in Montanide ISA51 and injected subcutaneously in combination with cyclophosphamide (300 mg/m(2)) and low-dose IL-2 (3 × 10(6) IU). The immune responses were monitored using ex vivo IFN-γ-ELISpot, HLA/multimer staining, and in vitro short-term peptide sensitization assays.

Results: Vaccination induced a rapid and persistent increase in specific effector memory CD8(+) T cells in 75% of the patients. However, this immunization was not associated with any significant increase in disease-free or overall survival as compared with the observation group. An extensive immunologic analysis revealed a significantly reduced cross-recognition of the corresponding native peptides and, most importantly, a limited ability to react to melanoma cells.

Conclusions: Adjuvant setting is an appealing approach for testing cancer vaccines because specific CD8(+) T cells can be efficiently induced in most vaccinated patients. However, the marginal antitumor activity of the T cells induced by modified peptides in this study largely accounts for the observed lack of benefit of vaccination. These findings suggest reconsidering this immunization strategy, particularly in early disease.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-1516DOI Listing
December 2012

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

Cancer Immunol Immunother 2007 Dec 13;56(12):1979-91. Epub 2007 Jun 13.

Unit of Immunotherapy of Human Tumors, IRCCS Istituto Nazionale Tumori, Via Venezian 1, 20133, Milan, Italy.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.
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http://dx.doi.org/10.1007/s00262-007-0342-zDOI Listing
December 2007

Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: role in immune escape.

Gastroenterology 2005 Jun;128(7):1796-804

Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milan, Italy.

Background & Aims: Normal and neoplastic cells release microvesicles, whose effects on the immune system still need to be elucidated. Because human colorectal cancer cells are hypothesized to escape immune recognition by expressing proapoptotic molecules, we investigated whether microvesicles bearing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand and inducing apoptosis of activated T cells are secreted by colorectal cancer cells both in vitro and in affected patients.

Methods: Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand expression were analyzed in colorectal cancer cells and purified microvesicles by flow cytometry, Western blotting, and immunoelectron microscopy. Microvesicle tumor origin was assessed through simultaneous detection of lysosomal (CD63) and adenocarcinoma (carcinoembryonic antigen) markers. Proapoptotic activity of microvesicles was evaluated by annexin V/propidium iodide staining and caspase activation in T cells, including CD8+ T lymphocytes from colorectal cancer patients.

Results: Colorectal cancer cells showed a granular pattern of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand expression, suggesting a secretory behavior. These proapoptotic molecules were detected on isolated microvesicles, together with class I HLA, CD63, and carcinoembryonic antigen. Microvesicles induced Fas ligand-mediated and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of activated CD8+ T cells generated from colorectal cancer patients. Microvesicles with comparable phenotypes and functions were found in plasma from patients with advanced disease, whereas vesicular structures expressing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand were also detected in colorectal cancer specimens.

Conclusions: These data show that colorectal cancer induces T-cell apoptosis through the release of Fas ligand-bearing and tumor necrosis factor-related apoptosis-inducing ligand-bearing microvesicles both in vitro and in vivo. This mechanism of immune escape has potential implications as a prognostic factor and could be targeted for the development of new antitumor therapies in colorectal cancer patients.
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http://dx.doi.org/10.1053/j.gastro.2005.03.045DOI Listing
June 2005

Natural killer and NK-Like T-cell activation in colorectal carcinoma patients treated with autologous tumor-derived heat shock protein 96.

Cancer Res 2005 May;65(9):3942-9

Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milan, Italy.

Heat shock proteins (HSPs) are involved in the activation of both adaptive and innate immune systems. Here, we report that vaccination with autologous tumor-derived HSP96 of colorectal cancer patients, radically resected for liver metastases, induced a significant boost of natural killer (NK) activity detected as cytokine secretion and cytotoxicity in the presence of NK-sensitive targets. Increased NK activity was associated with a raise in CD3-CD56+ NK and/or CD3+CD56+ NK-like T cells, displaying enhanced expression of NKG2D and/or NKp46 receptors. Up-regulated expression of CD83 and CD40 and increased interleukin-12 release on stimulation were observed in CD14+ cells from post-HSP96 peripheral blood mononuclear cells, suggesting an indirect pathway of NK stimulation by HSP96-activated monocytes. Additionally, CD3-CD56+ and CD3+CD56+ lymphocytes were found to undergo functional and phenotypic activation on in vitro exposure to HSP96 even in the absence of monocytes, supporting a potential direct activity of HSP96 on these cell subsets. This evidence was confirmed by the specific binding of FITC-conjugated HSP96 to a subset of both CD3-CD56+ and CD3+CD56+ cells in peripheral blood mononuclear cells from colorectal cancer patients. Altogether, these findings identify the activation of the NK compartment as an additional immunologic effect of autologous tumor-derived HSP96 administration in cancer patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-3493DOI Listing
May 2005

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells.

J Immunol 2003 Oct;171(7):3467-74

Unit of Immunotherapy of Human Tumor, Gastrointestinal and Liver Surgery Unit, and. Colorectal Surgery Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8(+) T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.
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http://dx.doi.org/10.4049/jimmunol.171.7.3467DOI Listing
October 2003

Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel, class II HLA-restricted melanoma antigen.

J Immunol 2003 Jun;170(12):6363-70

Unit of Immunotherapy of Human Tumors and Unit of Molecular Mechanisms of Tumor Growth and Progression, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.
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http://dx.doi.org/10.4049/jimmunol.170.12.6363DOI Listing
June 2003

Suboptimal activation of CD8(+) T cells by melanoma-derived altered peptide ligands: role of Melan-A/MART-1 optimized analogues.

Cancer Res 2003 Apr;63(7):1560-7

Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, 20133 Milan, Italy.

Suboptimal activation of T lymphocytes by tumor cells may contribute to the failure of the immune system to control tumor growth. We recently demonstrated that Melan-A/MART-1-reactive CTLs can be anergized by peptide analogues with partial agonist/antagonist functions, which selectively impair interleukin (IL)-2 release. Here we analyze the potential expression of partial agonist/antagonist peptides by tumor cells and their role in suboptimal T-cell activation. HLA-bound peptide fractions were eluted from HLA-A*0201/Melan-A/MART-1(+) melanoma cells and analyzed for reconstitution of the MART-1-specific T-cell epitope. Among the peptide fractions able to induce IFN-gamma release by MART-1-specific T cells, only fraction 43-44 activated IL-2 production by anti-MART-1 T cells, whereas the remaining two fractions acted as peptide antagonists by inhibiting IL-2 release in response to the native epitope. A comparable down-modulation of IL-2 release could also be induced by the MART-1-derived peptide 32-40, previously identified in one of the two anergizing fractions. A substantial deficit in IL-2 release was additionally detected in tumor-specific CD8(+) T cells infiltrating melanoma lesions. To overcome IL-2 impairment by peptide antagonists, anti-MART-1 T cells were generated by in vitro sensitization with the two optimized analogues Melan-A/MART-1(27-35) 1L (with superagonist features) and Melan-A/MART-1(26-35) 2L (with improved HLA-A*0201 binding). T cells raised with the superagonist Melan-A/MART-1(27-35) 1L showed resistance to the inhibition of IL-2 release mediated by melanoma-derived peptide fractions, whereas Melan-A/MART-1(26-35) 2L-specific T cells appeared to be as sensitive as T cells raised with the parental epitope. This resistance was associated with the enhanced ability of Melan-A/MART-1(27-35) 1L-specific T cells to release IL-2. Taken together, these data indicate that melanoma cells can process and present on their surface peptides inhibiting optimal T-cell activation against immunodominant epitopes and that the usage of optimized peptide analogues could represent a promising approach for overcoming tumor-induced immunosuppression and possibly designing more successful vaccines for cancer patients.
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April 2003

Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles.

J Exp Med 2002 May;195(10):1303-16

Unit of Immunotherapy of Human Tumors, Istituto Nazionale dei Tumori, Milan 20133, Italy.

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as 'Fas tumor counterattack,' has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2193755PMC
http://dx.doi.org/10.1084/jem.20011624DOI Listing
May 2002