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Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed “early effector cells” (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non–small cell lung cancer. Cancer Res; 77(4); 851–61. ©2016 AACR.

CD8+ T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8+ T cells expressing FOXP3. CD8+ FOXP3+ CD25+ T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8+ FOXP3+ T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-β1], exhausted (PD-1), or senescent (CD57) CD8+ T lymphocytes. Instead, this subset showed an antigen-experienced “EM1” phenotype (CCR7− CD45RA− CD28+ CD27+) and exhibited a CD127−, KLRG1−, HLA-DR+, CD38+, T-bet+, perforin+ “early effector” profile predicted by current models. CD8+ FOXP3+ T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8+ FOXP3+ T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8+ FOXP3+ “early effector” subset may be an invaluable tool for monitoring immunity at tumor site. Cancer Res; 70(21); 8378–87. ©2010 AACR.

Introduction Tumor microenvironment plays an essential role in cancer development and progression. In the context of thyroid cancer (TC) it still represents a poorly investigated field. Recent data from in vitro and in vivo studies proposed a model of TC progression promoted by the converging action of thyroid tumor cells and cancer-associated fibroblasts (CAFs). According to this mouse model CAFs are recruited in tumor stroma where synthesize and deposit collagen (COL1A1) that is, in turn, cross-linked by the enzyme LOX, produced by the thyroid tumor cells. Collectively this coordinated action lead to matrix stiffness and TC progression. Interestingly, this process is reported to be specifically observed in murine thyroid tumors expressing BRAFV600E mutation, but not in those harboring RAS mutation. In this study we tested this cross-talk in human thyroid cancer tissues. Experimental procedures A series of thyroid specimens, comprising different tumor subtypes (PTC and PDTC) and histologic variants, was collected at our institution and investigated by immunohistochemical (IHC) analyses for the expression of α-SMA (marker for activated fibroblasts), collagen COL1A1 and LOX. Tumor samples were also screened for the most common genetic lesions reported in TC including BRAFV600E and N/H/KRAS mutations. Publicly available datasets of thyroid tumors and non-neoplastic thyroid controls, analyzed on Affymetrix HG-U133 Plus 2.0 array, were selected to investigate the expression profiles of α-SMA, COL1A1 and LOX genes. Robust Multi Array Average (RMA) normalization, Brain Array annotation and batch correction procedures were applied to obtain gene expression profiles across all samples. Results In IHC analyses α-SMA decoration was observed in the stroma of thyroid tumor tissues while was restricted to blood vessels in the adjacent non-neoplastic thyroid. Stromal α-SMA staining was not evenly distributed in the tumors but localized preferentially at the tumor invasive border, organized in peripheral structures as fibrous septa or capsule. In agreement with the proposed model, higher level of α-SMA staining were specifically observed in PTC tumors harboring BRAFV600E mutation compared with tumors harboring RAS mutation. IHC analyses on tissue serial sections confirmed the coordinated expression of α-SMA, COL1A1 and LOX; COL1A1 localized in tumor stroma and was closely correlated with α-SMA positive areas while LOX was expressed by thyroid tumor cells. The coordinated expression of the three genes was confirmed by meta-analysis of public datasets. Conclusions In this study we provide evidence of the coordinated presence of thyroid tumor cells, activated fibroblasts and proteins of extracellular matrix, such as COL1A1 and LOX, in the contest of human thyroid carcinoma. Citation Format: Emanuela Minna, Silvia Brich, Katia Todoerti, Silvana Pilotti, Federica Perrone, Nicholas Paielli, Luca Agnelli, Ilaria Bersani, Roberta Mortarini, Andrea Anichini, Paola Collini, Antonino Neri, Angela Greco, Maria Grazia Borrello. Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4071.

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1–dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1− cells) nor up-regulated (in APAF-1+ cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1+ and APAF-1− tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 –specific inhibitors in both APAF-1+ and APAF-1− tumor cells. In response to 1 to 100 μmol/L of cisplatin, camptothecin, or celecoxib, APAF-1+ melanomas (n = 12) did not show significantly increased levels of apoptosis compared with APAF-1− tumors (n = 7), with the exception of enhanced apoptosis in response to a very high dose (100 μmol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.

Activation of CTL-mediated antitumor immunity to self-epitopes expressed by neoplastic cells is thought to be prevented, at any stage of tumor progression, by tolerance mechanisms. In contrast, in 74 American Joint Committee on Cancer stages I-IV melanoma patients, we found that development of lymph node metastases is a key event triggering CD8(+) T-cell-mediated immunity to self-epitopes encoded by melanocyte differentiation antigens. This was shown by the increased peripheral precursor frequency to Melan-A/Mart-1, gp100, and tyrosinase epitopes in stage III and IV compared with stage I and II patients, and by accumulation of functional memory T cells directed to Melan-A/Mart-1(26-35) in tumor-invaded lymph nodes. However, in tumor-invaded lymph nodes of most patients, CD8(+) T cells directed to melanocyte differentiation antigens or to tumor-restricted antigens (MAGE-3 and NY-ESO-1 epitopes), showed a CCR7(+) CD45RA(+) CD27(+) CD28(+) perforin(-) "precursor" phenotype. Only in 7 of 23 cases antigen-specific CD8(+) T cells in invaded lymph nodes showed a predominant CCR7(-) CD45RA(-) CD27(+) CD28(-) perforin(+) "preterminally differentiated" phenotype. In the latter subset of patients, by immunohistochemistry in lymph node lesions, we found that CD8(+) T lymphocytes intermingling with the neoplastic tissue expressed a CCR7(-) CD45RO(+)/RA(-) phenotype, whereas CD4(+) lymphocytes did not infiltrate the tumor. Furthermore, perforin and granzyme B were expressed on a higher fraction of the CD8(+) cells surrounding the invading tumor compared with the lymphocytes infiltrating the neoplastic tissue. In addition, no evidence for tumor regression was found in such metastatic lesions, as documented by absence of neoplastic cell necrosis or apoptosis. These data indicate that neoplastic cells in the lymph nodes and/or increased tumor burden in metastatic disease activate CD8(+) T-cell-mediated antitumor immunity to self-epitopes. However, the paucity of terminally differentiated CD8(+) T cells at tumor site suggests that immunotherapy strategies may require not only the boosting of tumor immunity, but also effective means to promote CD8(+) T-cell differentiation in the neoplastic tissue.

Focal adhesion kinase (FAK) is a tyrosine kinase localized at the site of focal adhesions (FA). FAK is identified as a key mediator of signaling by integrins, the major family of cell surface receptors for extracellular matrix, as well as by other receptors in both normal and cancer cells. FAK plays a prominent role in tumor progression and metastasis regulating cellular processes including migration, invasion, epithelial to mesenchymal transition, and angiogenesis. Integrin-FAK signaling has been shown to activate a number of biological mechanisms through phosphorylation and protein-protein interactions promoting tumorigenesis. FAK seems also to have a role in the proliferation of tumor cells by its biochemical and biological association to cytoplasmic kinases such as src and ERK. However, the mechanisms that involve FAK in those processes are not yet clarify. The best characterized FAK phosporylation event is the auto-phosphorylation at the tyrosine397 (Tyr397) which creates a motif that is recognized by various SH2 domain-containing proteins, such as src which phosphorylates FAK on Tyr576. The c-terminal domain of FAK undergoes to several serine-phosphorylation events whose role is not well-understood. Previous studies in neural and endothelial cells (EC) have shown a new role for FAK in the regulation of centrosome functions during mitosis dependent on Ser732 phosphorylation. We have found that EGF stimulation of selected starved melanoma, thyroid and high stage ovarian tumor cells induced, in dividing cells, accumulation of phosphorylated (P-)FAK on Ser732. Inhibition of cell/substrate adhesion with specific anti-integrin antibodies demonstrated that P-FAKSer732 activation is not induced by integrin clustering. Confocal immunofluorescence and immunoprecipitation showed that, in these cells, P-FAKSer732 is localized in a perinuclear site independently from FA and P-FAKTyr397. Treatment of an high proliferative melanoma cell line with the Rock inhibitor Y27632 partially decreased the levels of P-FAKSer732, the cellular proliferation and impaired the mitotic spindle formation. FACS analysis showed that the P-FAKSer732 decreased upon treatment with the MEK inhibitor UO126 in a dose-dependent manner together with the levels of P(Ser 10) -Histone H3 thus demonstrating that FAK is involved in the mitotic process regulated by MEK/ERK activation. Indeed, P-FAKSer732 co-localized with microtubules of the mitotic spindle in proliferating cells and immunoprecipitated with the motor protein dynein and with the acetylated tubulin of polymerized microtubules. The finding of a novel role for FAK in tumor cells might provide the underpinning for therapeutic strategies in selected solid tumors. Partially supported by Italian Ministery of Health and Associazione Italiana per la Ricerca sul Cancro (AIRC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 226. doi:1538-7445.AM2012-226