Supplementary Figure S4. Characterization of additional cell lines. A. Immunofluorescence for DAPI (blue) and CGA (red) on murine NEPC cells (TC566 and TC411K). B. Immunofluorescence for DAPI (blue) and OPN (red) on HMC-1 cells. C. Western blot for OPN in HMC-1 cells treated or not with Brefeldin A (BFA; 5 g/ml). Vinculin was evaluated as housekeeping control. D. Gating strategy applied to distinguish human NEPC cells (CD49f+c-Kit-) from HMC-1 MCs (CD49f-c-Kit+) by flow cytometry. E. OPN evaluation by ELISA (left panel) or real time PCR (the Spp1 transcript; right panel) in WT and OPN−/− MCs, and in MC/9, T23, and ST4787 cells. F. Elisa for OPN in parental MC/9 cells and in MC/9-CTR, MC/9-OPNf, MC/9-iOPN cells. T23 cells were used as control.

Supplementary table 1 - list of antibodies used in the study

Supplementary Figure S1. Flow cytometry evaluation of murine MC purity and maturation in vitro. A. Gating strategy used to evaluate the purity and maturation of WT, OPN-/-, MyD88-/- and TNFa-/-MCs by flow cytometry. Mature MC population is identified as c-Kit+FceRI+.

Supplementary Figure S5. Expression of OPN in MC/9 cells and effect on T23 cell proliferation. A. Separate channels of immunofluorescence for DAPI (cyan), OPN (red) and WGA (blue) of MC/9, MC/9-CTR, MC/9-OPNf, and MC/9-iOPN cells showed in Fig. 2E. B. Quantification of A as percentage of OPN positive cells. C. Murine adenocarcinoma (T23) cells (50.000/well) were cultured either alone or with MC/9, MC/9-CTR, MC/9-OPNf or MC/9-iOPN cells (tumor cell:MC ratio 1:1). After 4 days the growth rate of cancer cells was evaluated through trypan blue count. Cancer cells and MC/9 could be distinguished thanks to their grown in adhesion or suspension, respectively. All histograms depict mean ± SD of biological replicates (represented by dots). One-way ANOVA followed by Tukey’s multiple comparison test was used: *, P < 0.05; **, P <0.01. Where P-value is not indicated, the comparison between groups is not statistically significant.

Supplementary table 3 - List of putative NEPC-specific surface TLR ligands

Supplementary Figure S2. OPN evaluation in bone marrow-derived MCs. A. Separate channels of immunofluorescence for DAPI (cyan), OPN (red) and WGA (blue) of WT and OPN-/- MCs showed in Fig. 1A B. Representative images of 3 different biological replicates of immunofluorescence for DAPI (cyan), OPN (red) and WGA (blue), in WT and OPN-/- MCs. These pictures were used for the quantification reported in Fig. 1B.

Supplementary Figure S8. Hematoxylin and eosin staining in TRAMP mice. A. Hematoxylin and eosin staining in serial slides of tumors reported in Fig. 5A, showing an untreated TRAMP mouse with adenocarcinoma (ADENO) and a TRAMP mouse subjected to surgical castration showing a focal t-NEPC area.

Supplementary table 4 - transcript levels of the 29 TLR ligands measured by high throughput customized Taqman assay

Supplementary Figure S9. Expression and silencing of putative TLR ligands in NEPC cells. A. Venn diagrams show a list of 29 genes (List 3; Supplementary Table S3) extrapolated from the intersection between genes found up-regulated in TRAMP-derived incipient NEPC (a data set generated in this study, GSE242811; List 1; Supplementary Table S3) and a list of surface TLR2/TLR4 ligands identified from a ligand-receptor pairs repository (LewisLabUCSD, ref. (Armingol et al., 2021) List 2; Table S3). B. Real Time-PCR for Cd14, Sdc1, Hspa2 and Anxa2 on lysates from T23 and ST4787 cells. Technical replicates are indicated by dots (n=2). C. Quantification of the western blot for HSPA2 and ANXA2 in T23 and ST4787 showed in Fig. 6B. D. Expression of CD14 and SDC1 in ST4787 silenced with two different siRNA specific for Cd14 (ST4787-siCD14- 1 and ST4787-siCD14-2) or Sdc1 (ST4787-siSDC1-1 and ST4787-siSDC1-2), or in scramble control cells (ST4787-scramble), evaluated by flow cytometry 72 hours post-silencing. E. HSPA2 and ANXA2 expression in ST4787 silenced with two different siRNA specific for Hspa2 (ST4787- siHSPA2-1 and ST4787- siHSPA2-2) or Anxa2 (ST4787-siANXA2-1 and ST4787-siANXA2-2), or in scramble control cells (ST4787-scramble) evaluated by western blot analysis 72 hours post- silencing. F. Quantification of E. Western blot was validated twice. G. ST4787-scramble or silenced with two different siRNA specific for Cd14 were cultured with WT or OPN−/− MCs (ratio 1:1). After 16 hours the percentage of TNF positive MCs was evaluated by intracellular flow cytometry in WT or OPN-/-MCs. Gating strategy is reported in Fig. S4D. H. Flow cytometry evaluation of CD14 on adenocarcinoma (22Rv1 human) and NEPC (TC411K murine, and NCI-H660 human) cells. All histograms depict mean ± SD of biological replicates (represented by dots). T test (B) or One-way ANOVA followed by Tukey’s multiple comparison test (G) were used for the analysis of significance between samples. P-values are reported as: *, P < 0.05; **, P <0.01; ***, P <0.001. Where P-value is not indicated, the comparison between groups is not statistically significant.

Supplementary table 2 - list of siRNA and Taqman assays used in the study

Supplementary Figure S3. Toluidine blue for detection of MCs in mice. A. MC count reported as number of MCs/tumor area (cm2) in tumor samples from TRAMP mice with prostate intraepithelial neoplasia or adenocarcinoma (PIN/ADENO, n= 12), or TRAMP mice with NEPC (n= 3), as well as in prostatectomies from untreated patients with Gleason Score 7 (n= 10), Gleason Score 8 (n=10), or showing evidence of NEPC (n= 4). The histogram depict mean ± SD of biological replicates (represented by dots). B. Upper panels: representative images of toluidine blue staining in TRAMP, KitWsh-TRAMP, and OPN-/-TRAMP mice, or in KitWsh-TRAMP mice reconstituted with WT, OPN-/- or TNFa-/- MCs. Lower panels: representative images of toluidine blue staining in a prostate of a patient with adenocarcinoma. Scale bars indicate magnifications.

Supplementary Figure S7. Flow cytometry characterization of TNFa receptors and TNFa production. A. List of cytokines and chemokines tested through a multiplex immunoassay (Procarta-plex by Thermofisher) in the supernatants of ST4787 or T23 cells cultured either alone or in the presence of WT, OPN-/-, or MyD88-/- MCs. B.-C. Flow cytometry evaluation of TNFRs (CD120a and CD120b) on adenocarcinoma (T23 murine, 22Rv1 human) and NEPC (TC566 and TC411K murine, and LASCPC-01 human) cells. D. Gating strategy applied for intracellular detection of TNFa in MCs (CD49f-CD45+) by flow cytometry, in the co-cultures between MCs and tumor cells.

Supplementary Figure S6. Quantification of western blot. A. Quantification of western blot reported in Fig. 3G. Western blots were validated twice. All histograms depict mean ± SD of biological replicates (represented by dots). One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis of significance between samples. P-values are reported as: *, P < 0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001. Where P-value is not indicated, the comparison between groups is not statistically significant.

Aim: Castration-resistant prostate cancer (CRPC) eventually becomes resistant to androgen receptor pathway inhibitors like enzalutamide. Immunotherapy also fails in CRPC. We propose a new approach to simultaneously revert enzalutamide resistance and rewire anti-tumor immunity. Methods: We investigated in vitro and in subcutaneous and spontaneous mouse models the effects of combining enzalutamide and GSK-126, a drug inhibiting the epigenetic modulator EZH2. Results: Enzalutamide and GSK-126 synergized to reduce CRPC growth, also restraining tumor neuroendocrine differentiation. The anti-tumor activity was lost in immunodeficient mice. Indeed, the combination treatment awoke cytotoxic activity and IFN-γ production of tumor-specific CD8+ T lymphocytes. Conclusion: These results promote the combination of enzalutamide and GSK-126 in CRPC, also offering new avenues for immunotherapy in prostate cancer.

Abstract Castration resistant prostate cancer (CRPC) is a fatal disease. Androgen receptor pathway inhibitors, like enzalutamide, are initially effective but resistance eventually occurs, often associated to the emergence of aggressive neuroendocrine variants (NEPC). Even immunotherapy induced limited results in prostate cancer, governed by an immunosuppressive microenvironment. Therefore, effective therapies are needed. We here investigate in preclinical models a new approach aimed to revert castration resistance and simultaneously turn the immune milieu from “cold” to “hot”, through the combination between enzalutamide and the drug GSK-126, which inhibits the epigenetic modulator EZH2. We show that enzalutamide and GSK-126 can synergize to restrain the growth of CRPC in vitro and in vivo. Moreover, this therapeutic combination efficiently reduced NEPC differentiation, in both subcutaneous and autochthonous in vivo models, increasing the rate of cured mice with regressed lesions. The antitumor efficacy observed in immunocompetent mice bearing subcutaneous syngeneic CRPC tumors was lost in immunodeficient mice, indicating a contribution of immune cells. Additional experiments in the TRAMP spontaneous mouse model revealed that the combination of enzalutamide and GSK-126 significantly induced cytotoxic activity and IFNγ production by tumor-specific CD8+ T cells, otherwise tolerant, and increased IL-17 production by CD4+ T cells. The two drugs did not directly modulate T cell activity in vitro, suggesting the importance of microenvironment accomplices in triggering these effects. These results promote the combined use of enzalutamide and GSK-126 to restrain CRPC growth and NEPC differentiation, and, simultaneously, to awake antitumor T cell response, opening new possibilities for immunotherapy in prostate cancer. Citation Format: Irene Fischetti, Botti Laura, Roberta Sulsenti, Valeria Cancila, Claudia Enriquez, Renata Ferri, Marco Bregni, Filippo Crivelli, Claudio Tripodo, Mario P. Colombo, Elena Jachetti. Combined therapy targeting AR and EZH2 restrains the growth of castration resistant prostate cancer by enhancing antitumor T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2639.

Abstract Neuroendocrine prostate cancer (NEPC) is an aggressive form of prostate cancer that emerges as tumors become resistant to hormone therapies or, rarely, arises de novo in treatment-naïve patients. The urgent need for effective therapies against NEPC is hampered by the limited knowledge of the biology governing this lethal disease. Based on our prior observations in the transgenic adenocarcinoma of the mouse prostate (TRAMP) spontaneous prostate cancer model, in which the genetic depletion of either mast cells (MC) or the matricellular protein osteopontin (OPN) increases NEPC frequency, we tested the hypothesis that MCs can restrain NEPC through OPN production, using in vitro co-cultures between murine or human tumor cell lines and MCs, and in vivo experiments. We unveiled a role for the intracellular isoform of OPN, so far neglected compared with the secreted isoform. Mechanistically, we unraveled that the intracellular isoform of OPN promotes TNFα production in MCs via the TLR2/TLR4-MyD88 axis, specifically triggered by the encounter with NEPC cells. We found that MC-derived TNFα, in turn, hampered the growth of NEPC. We then identified the protein syndecan-1 (SDC1) as the NEPC-specific TLR2/TLR4 ligand that triggered this pathway. Interrogating published single-cell RNA-sequencing data, we validated this mechanism in a different mouse model. Translational relevance of the results was provided by in silico analyses of available human NEPC datasets and by immunofluorescence on patient-derived adenocarcinoma and NEPC lesions. Overall, our results show that MCs actively inhibit NEPC, paving the way for innovative MC-based therapies for this fatal tumor. We also highlight SDC1 as a potential biomarker for incipient NEPC.

Primary cutaneous gamma delta T-cell lymphomas (PCGDTLs) are a heterogeneous group of lymphomas representing about 1% of primary cutaneous T-cell lymphomas (CTCLs) and mostly regarded as clinically aggressive. Current WHO-EORTC classification recognizes different clinic-pathologic subsets of PCGDTL, but it suggests that cases showing a mycosis fungoides (MF)-like clinical presentation and histopathology should be classified as MF irrespective of phenotype for their indolent course. Herein, we describe a case of gamma delta-MF, featuring at onset a granulomatous pattern, with subsequent clinical worsening signaled by the development of an ulceronecrotic lesion and systemic dissemination, leading to death in 5 months. Clinical progression was sustained by a shift to mature T-cell lymphoma composed of medium to large-sized blastoid T-cells featuring a t-cell receptor (TCR) silent immunophenotype.

The COVID-19 (Coronavirus Disease-19) is the most urgent health emergency worldwide and all professionals are called to give support in the diagnosis and treatment of patients affected by this disease. The Scientific Society of Hospital Legal Medicine of the National Health System (COMLAS) and the Italian Society of Anatomical Pathology and Cytology (SIAPEC) produced this document with the intent of offering a technical support to professional involved in the autoptic activities during the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) epidemic infection.

The World Health Organization (WHO) classification of hematopoietic and lymphoid tumors identifies distinctive subtypes of peripheral T-cell lymphoma (PTCL), and, additionally, some PTCLs involving mostly extranodal sites like the skin. The difficulty of classifying PTCLs according to the normal stages of T-cell differentiation and the lack of definitive diagnostic markers for most of the subtypes make the diagnosis of these diseases challenging. PTCL cases which do not fit into any of the specifically defined entities are categorized as PTCL not otherwise specified (PTCL-NOS). PTCLs-NOS represent less than 2% of the total cases of T-cell lymphoma involving the skin. This article illustrates a case of a PTCL-NOS in which tumor cells have an activated cytotoxic TCRαβ+CD3+CD4+CD56+ T-cell phenotype and histopathologic features of subcutaneous panniculitis-like T-cell lymphoma, leading to a fatal outcome.

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p < 0.05).The HERV env gene was expressed in the EVs at of 54% (-H), 38% (-K), 31% (-R) patients. Association was not found between HERV env expression and LTR methylation, but significant higher expression of HERV-P and -R env was found in tumor tissues arising from the right colon. Our findings do not demonstrate significant overexpression of the studied HERV in colorectal cancer, but their association with tumor localization and specificity of the changes in DNA methylation of retroelements are shown. HERV sequences were packaged in the EVs and might be transferred from one cell to another.