Applied immunohistochemistry & molecular morphology : AIMM
Miller, CP;Shokri, F;Akilesh, S;Xu, Y;Warren, EH;Tykodi, SS;Tretiakova, M;
PMID: 36735485 | DOI: 10.1097/PAI.0000000000001101
5T4 (trophoblast glycoprotein encoded by TPBG) is a cancer/testis antigen highly expressed in renal cell carcinoma (RCC) and many other cancers but rarely in normal tissues. Interest in developing 5T4 as a prognostic biomarker and direct targeting of 5T4 by emerging receptor-engineered cellular immunotherapies has been hampered by the lack of validated 5T4-specific reagents for immunohistochemistry (IHC). We tested 4 commercially available monoclonal antibodies (mAbs) for the detection of 5T4 in formalin-fixed, paraffin-embedded RCC and normal tissues. Using parental and TPBG-edited A498 cells, 3 mAbs showed 5T4 specificity. Further analyses focused on 2 mAbs with the most robust staining (MBS1750093, Ab134162). IHC on tissue microarrays incorporating 263 renal tumors showed high staining concordance of these 2 mAbs ranging from 0.80 in chromophobe RCC to 0.89 in advanced clear cell RCC (ccRCC). MBS1750093, the most sensitive, exhibited 2+/3+ staining in papillary RCC (92.2%) > advanced ccRCC (60.0%) > chromophobe RCC (43.6%) > localized ccRCC (39.6%) > oncocytoma (22.7%). RNA in situ hybridization also revealed high levels of TPBG RNA were present most frequently in papillary and advanced ccRCC. In advanced ccRCC, there was a trend towards higher 5T4 expression and regional or distant metastases. Normal organ controls showed no or weak staining with the exception of focal moderate staining in kidney glomeruli and distal tubules by IHC. These data identify mAbs suitable for detecting 5T4 in formalin-fixed, paraffin-embedded tissues and demonstrate both interpatient and histologic subtype heterogeneity. Our validated 5T4 IHC protocol will facilitate biomarker studies and support the therapeutic targeting of 5T4.
Annals of Gastroenterological Surgery
Ichihara, M;Takahashi, H;Nishida, N;Ivan, C;Okuzaki, D;Yokoyama, Y;Ohtsuka, M;Miyoshi, N;Uemura, M;Tanaka, S;Calin, G;Mori, M;Doki, Y;Eguchi, H;Yamamoto, H;
| DOI: 10.1002/ags3.12649
Background Studies have shown that cancer stemness and the endoplasmic reticulum (ER) stress response are inversely regulated in colorectal cancer (CRC), but the mechanism has not been fully clarified. Long noncoding RNAs (lncRNAs) play key roles in cancer progression and metastasis. In this study we investigated lncRNA 01534 (LINC01534) as a possible modulator between cancer stemness and ER stress response. Methods In vitro experiments using CRC cell lines were performed to explore a possible role of LINC01534. The expression of LINC01534 in clinical CRC samples was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization. Results Silencing LINC01534 led to suppression of cell proliferation, invasiveness, and cell cycle progression at the G2-M phase, and promoted apoptosis. Moreover, we found that silencing LINC01534 suppressed cancer stemness, while it activated the ER stress response, especially through the PERK/eIF2α signaling pathway. In situ hybridization revealed LINC01534 was expressed in tumor cells and upregulated in CRC tissues compared with normal epithelium. A survival survey indicated that high LINC01534 expression was significantly associated with shorter overall survival in 187 CRC patients. Conclusion This is the first report on LINC01534 in human cancer. Our findings suggest that LINC01534 may be an important modulator of the maintenance of cancer stemness and suppression of the ER stress response, and that it could be a novel prognostic factor in CRC.
Cellular and molecular life sciences : CMLS
Gillot, L;Lebeau, A;Baudin, L;Pottier, C;Louis, T;Durré, T;Longuespée, R;Mazzucchelli, G;Nizet, C;Blacher, S;Kridelka, F;Noël, A;
PMID: 35567669 | DOI: 10.1007/s00018-022-04262-w
Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.
Lecker, LSM;Berlato, C;Maniati, E;Delaine-Smith, R;Pearce, OMT;Heath, O;Nichols, SJ;Trevisan, C;Novak, M;McDermott, J;Brenton, JD;Cutillas, PR;Rajeeve, V;Hennino, A;Drapkin, R;Loessner, D;Balkwill, FR;
PMID: 34561272 | DOI: 10.1158/0008-5472.CAN-21-0536
The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as βig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFβ and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvβ3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated β3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFβ and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.
Prognostic Value of PD-L1, PD-1 and CD8A in Canine Diffuse Large B-Cell Lymphoma Detected by RNAscope
Aresu, L;Marconato, L;Martini, V;Fanelli, A;Licenziato, L;Foiani, G;Melchiotti, E;Nicoletti, A;Vascellari, M;
PMID: 34209830 | DOI: 10.3390/vetsci8070120
Immune checkpoints are a set of molecules dysregulated in several human and canine cancers and aberrations of the PD-1/PD-L1 axis are often correlated with a worse prognosis. To gain an insight into the role of immune checkpoints in canine diffuse large B-cell lymphoma (cDLBCL), we investigated PD-L1, PD-1 and CD8A expression by RNAscope. Results were correlated with several clinico-pathological features, including treatment, Ki67 index and outcome. A total of 33 dogs treated with chemotherapy (n = 12) or chemoimmunotherapy with APAVAC (n = 21) were included. PD-L1 signal was diffusely distributed among neoplastic cells, whereas PD-1 and CD8A were localized in tumor infiltrating lymphocytes. However, PD-1 mRNA was also retrieved in tumor cells. An association between PD-L1 and PD-1 scores was identified and a higher risk of relapse and lymphoma-related death was found in dogs treated with chemotherapy alone and dogs with higher PD-L1 and PD-1 scores. The correlation between PD-L1 and PD-1 is in line with the mechanism of immune checkpoints in cancers, where neoplastic cells overexpress PD-L1 that, in turn, binds PD-1 receptors in activated TIL. We also found that Ki67 index was significantly increased in dogs with the highest PD-L1 and PD-1 scores, indirectly suggesting a role in promoting tumor proliferation. Finally, even if the biological consequence of PD-1+ tumor cells is unknown, our findings suggest that PD-1 intrinsic expression in cDLBCL might contribute to tumor growth escaping adaptive immunity.
miR-31 Displays Subtype Specificity in Lung Cancer
Davenport, ML;Echols, JB;Silva, AD;Anderson, JC;Owens, P;Yates, C;Wei, Q;Harada, S;Hurst, DR;Edmonds, MD;
PMID: 33558335 | DOI: 10.1158/0008-5472.CAN-20-2769
miRNA rarely possess pan-oncogenic or tumor-suppressive properties. Most miRNAs function under tissue-specific contexts, acting as either tumor suppressors in one tissue, promoting oncogenesis in another, or having no apparent role in the regulation of processes associated with the hallmarks of cancer. What has been less clear is the role of miRNAs within cell types of the same tissue and the ability within each cell type to contribute to oncogenesis. In this study, we characterize the role of one such tissue-specific miRNA, miR-31, recently identified as the most oncogenic miRNA in lung adenocarcinoma, across the histologic spectrum of human lung cancer. Compared with normal lung tissue, miR-31 was overexpressed in patient lung adenocarcinoma, squamous cell carcinoma, and large-cell neuroendocrine carcinoma, but not small-cell carcinoma or carcinoids. miR-31 promoted tumor growth in mice of xenografted human adenocarcinoma and squamous cell carcinoma cell lines, but not in large- or small-cell carcinoma lines. While miR-31 did not promote primary tumor growth of large- and small-cell carcinoma, it did promote spontaneous metastasis. Mechanistically, miR-31 altered distinct cellular signaling programs within each histologic subtype, resulting in distinct phenotypic differences. This is the first report distinguishing diverse functional roles for this miRNA across the spectrum of lung cancers and suggests that miR-31 has broad clinical value in human lung malignancy. SIGNIFICANCE: These findings demonstrate the oncogenic properties of miR-31 in specific subtypes of lung cancer and highlight it as a potential therapeutic target in these subtypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/1942/F1.large.jpg.
Cancer Immunology Research
Wing A, Fajardo CA, Posey AD, Shaw C, Da T, Young R, Alemany R, June CH, Guedan S.
PMID: 29588319 | DOI: 10.1158/2326-6066.CIR-17-0314
T cells expressing chimeric antigen receptors (CART) have shown significant promise in clinical trials to treat hematologic malignancies, but their efficacy in solid tumors has been limited. Oncolytic viruses have the potential to act in synergy with immunotherapies due to their immunogenic oncolytic properties and the opportunity of incorporating therapeutic transgenes in their genomes. Here, we hypothesized that an oncolytic adenovirus armed with an EGFR-targeting, bispecific T-cell engager (OAd-BiTE) would improve the outcome of CART-cell therapy in solid tumors. We report that CART cells targeting the folate receptor alpha (FR-α) successfully infiltrated preestablished xenograft tumors but failed to induce complete responses, presumably due to the presence of antigen-negative cancer cells. We demonstrated that OAd-BiTE-mediated oncolysis significantly improved CART-cell activation and proliferation, while increasing cytokine production and cytotoxicity, and showed an in vitro favorable safety profile compared with EGFR-targeting CARTs. BiTEs secreted from infected cells redirected CART cells toward EGFR in the absence of FR-α, thereby addressing tumor heterogeneity. BiTE secretion also redirected CAR-negative, nonspecific T cells found in CART-cell preparations toward tumor cells. The combinatorial approach improved antitumor efficacy and prolonged survival in mouse models of cancer when compared with the monotherapies, and this was the result of an increased BiTE-mediated T-cell activation in tumors. Overall, these results demonstrated that the combination of a BiTE-expressing oncolytic virus with adoptive CART-cell therapy overcomes key limitations of CART cells and BiTEs as monotherapies in solid tumors and encourage its further evaluation in human trials.
Hong M, Lee T, Young Kang S, Kim SJ, Kim W, Ko YH.
PMID: 27015135 | DOI: 10.1038/modpathol.2016.47
Extranodal natural killer (NK)/T-cell lymphoma (ENKTL), nasal type, comprises NK or cytotoxic T cells. We evaluated the clinical impact of cell type and the usefulness of T-cell receptor (TCR) gene transcripts in distinguishing cell lineage. One hundred and eight cases of ENKTL were analyzed for TCR gene rearrangements using the BIOMED-2 protocol and for TCR gene expression using immunohistochemistry for TCR-βF1 and TCR-cγM1, and RNA in situ hybridization for TCR gene transcripts. Prognostic factors were analyzed. Among the 108 cases, 44 were monoclonal for a TCR rearrangement (40%) while 64 (60%) were undefinable. The monoclonal cases expressed TCR-βF1 in 14 out of 40 cases (35%) and TCR-cγM1 in 1 out of 44 cases (2%). The 64 undetermined cases expressed TCR-βF1 in 15 cases (23%) and TCR-cγM1 in 1 (2%). Thirteen of 40 TCR-β constant gene transcript-positive cases (33%) expressed TCR-βF1 and one of nine TCR-γ constant gene transcript-positive cases (11%) expressed TCR-cγM1. TCR gene transcripts were not useful in the distinction of cell lineages. TCR gene transcripts were positive in ENKTLs as well as in normal B cells and aggressive NK-cell leukemia. Based on gene rearrangements and immunohistochemistry for TCR, there were 60 T-cell type cases (56%), 32 NK-cell type cases (30%), and 16 cases with an undetermined cell type (14%). TCR protein was expressed in 30/60 T-ENKTLs (50%) in a variable fraction of tumor cells. There were no significant differences in clinical findings or overall patient survival between T- or NK-cell types of ENKTL, although those with a T-cell type tended to show a better prognosis for those with localized nasal lymphomas. Univariate and multivariate analysis showed that a non-nasal ENKTL, age >60 years, high level of lactate dehydrogenase, bone marrow involvement, and the absence of radiotherapy were independent prognostic factors.
Hilkens J, Timmer NC, Boer M, Ikink GJ, Schewe M, Sacchetti A, Koppens MA, Song JY, Bakker ER.
PMID: 27511199 | DOI: 10.1136/gutjnl-2016-311606
Abstract
OBJECTIVE:
The gross majority of colorectal cancer cases results from aberrant Wnt/β-catenin signalling through adenomatous polyposis coli (APC) or CTNNB1 mutations. However, a subset of human colon tumours harbour, mutually exclusive with APC and CTNNB1 mutations, gene fusions in RSPO2 or RSPO3, leading to enhanced expression of these R-spondin genes. This suggested that RSPO activation can substitute for the most common mutations as an alternative driver for intestinal cancer. Involvement of RSPO3 in tumour growth was recently shown in RSPO3-fusion-positive xenograft models. The current study determines the extent into which solely a gain in RSPO3 actually functions as a driver of intestinal cancer in a direct, causal fashion, and addresses the in vivo activities of RSPO3 in parallel.
DESIGN:
We generated a conditional Rspo3 transgenic mouse model in which the Rspo3 transgene is expressed upon Cre activity. Cre is provided by cross-breeding with Lgr5-GFP-CreERT2 mice.
RESULTS:
Upon in vivo Rspo3 expression, mice rapidly developed extensive hyperplastic, adenomatous and adenocarcinomatous lesions throughout the intestine. RSPO3 induced the expansion of Lgr5+ stem cells, Paneth cells, non-Paneth cell label-retaining cells and Lgr4+ cells, thus promoting both intestinal stem cell and niche compartments. Wnt/β-catenin signalling was modestly increased upon Rspo3 expression and mutant Kras synergised with Rspo3 in hyperplastic growth.
CONCLUSIONS:
We provide in vivo evidence that RSPO3 stimulates the crypt stem cell and niche compartments and drives rapid intestinal tumorigenesis. This establishes RSPO3 as a potent driver of intestinal cancer and proposes RSPO3 as a candidate target for therapy in patients with colorectal cancer harbouring RSPO3 fusions.
Verhoef EI, Kolijn K, De Herdt MJ, van der Steen B, Hoogland AM, Sleddens HF, Looijenga LH, van Leenders GJ.
PMID: 27105539 | DOI: 10.18632/oncotarget.8829
Tyrosine-kinase inhibitors of the hepatocyte growth factor receptor MET are under investigation for the treatment of hormone-refractory prostate cancer (HRPC) metastasis. Analysis of MET protein expression and genetic alterations might contribute to therapeutic stratification of prostate cancer patients. Our objective was to investigate MET on protein, DNA and RNA level in clinical prostate cancer at various stages of progression. Expression of MET was analyzed in hormone-naive primary prostate cancers (N=481), lymph node (N=40) and bone (N=8) metastases, as well as HRPC (N=54) and bone metastases (N=15). MET protein expression was analyzed by immunohistochemistry (D1C2 C-terminal antibody). MET mRNA levels and MET DNA copy numbers were determined by in situ hybridization. None of the hormone-naive primary prostate cancer or lymph node metastases demonstrated MET protein or mRNA expression. In contrast, MET protein was expressed in 12/52 (23%) evaluable HRPC resections. RNA in situ demonstrated cytoplasmic signals in 14/54 (26%) of the HRPC patients, and was associated with MET protein expression (p=0.025, χ2), in absence of MET amplification or polysomy. MET protein expression was present in 7/8 (88%) hormone-naive and 10/15 (67%) HRPC bone metastases, without association of HRPC (p=0.37; χ2), with MET polysomy in 8/13 (61%) evaluable cases. In conclusion, MET was almost exclusively expressed in HRPC and prostate cancer bone metastasis, but was not related to MET amplification or polysomy. Evaluation of MET status could be relevant for therapeutic stratification of late stage prostate cancer.
Opoku-Acheampong AB, Henningson JN, Beck AP, Lindshield BL.
PMID: 28493878 | DOI: 10.1371/journal.pone.0175874
Abstract
BACKGROUND:
The contribution of 5α-reductase 1 and 5α-reductase 2 to prostate cancer development and progression is not clearly understood. TRAMP mice are a common prostate cancer model, in which 5α-reductase 1 and 5α-reductase 2 expression levels, along with prostate lesions scores, have not been investigated at different time points to further understand prostate carcinogenesis.
METHOD/PRINCIPAL FINDINGS:
To this end, 8-, 12-, 16-, and 20-week-old male C57BL/6TRAMP x FVB mice prostate most severe and most common lesion scores, 5α-reductase 1 and 5α-reductase 2 in situ hybridization expression, and Ki-67, androgen receptor, and apoptosis immunohistochemistry levels were measured. Levels of these markers were quantified in prostate epithelium, hyperplasia, and tumors sections. Mice developed low- to high-grade prostatic intraepithelial neoplasia at 8 weeks as the most severe and most common lesions, and moderate- and high-grade prostatic intraepithelial neoplasia at 12 and 16 weeks as the most severe lesion in all lobes. Moderately differentiated adenocarcinoma was observed at 20 weeks in all lobes. Poorly differentiated carcinoma was not observed in any lobe until 12-weeks-old. 5α-reductase 1 and 5α-reductase 2 were not significantly decreased in tumors compared to prostate epithelium and hyperplasia in all groups, while proliferation, apoptosis, and androgen receptor were either notably or significantly decreased in tumors compared with prostate epithelium and hyperplasia in most or all groups. Prostate 5αR1 levels were positively correlated with adjusted prostate most severe lesion scores.
CONCLUSION:
Downregulation of androgen receptor and 5α-reductase 2, along with upregulation of 5α-reductase 1 in tumors may promote prostatic intraepithelial neoplasia and prostate cancer development in TRAMP mice.
Xiao G, Yao J, Kong D, Ye C, Chen R, Li L, Zeng T, Wang L, Zhang W, Shi X, Zhou T, Li J, Wang Y, Xu CL, Jiang J, Sun Y.
| DOI: 10.1016/j.eururo.2018.11.012
Abstract Background The link between prostate cancer (PCa) development and aberrant expression of genes located on the Y chromosome remains unclear. Objective To identify Y-chromosomal long noncoding RNAs (lncRNAs) with critical roles in PCa and to clarify the corresponding mechanisms. Design, setting, and participants Aberrantly expressed lncRNAs on the Y chromosome were identified using transcriptome analysis of PCa clinical samples and cell lines. Biological functions and molecular mechanisms of the lncRNAs were revealed using in vitro and in vivo experimental methods. Outcome measurements and statistical analysis Experiments and outcome measurements were performed in duplicate or triplicate. Wilcoxon signed-rank test was employed for comparison of RNA levels in clinical cohorts. Analysis of variance was employed for comparisons among multiple groups. Results and limitations In most patients with PCa, TTTY15 was the most elevated lncRNA located on the Y chromosome. Knockout of this lncRNA by two different CRISPR-Cas9 strategies suppressed PCa cell growth both in vitro and in vivo. TTTY15 promoted PCa by sponging the microRNA let-7, consequently increasing CDK6 and FN1 expression. FOXA1 is an upstream regulatory factor of TTTY15 transcription. Conclusions The Y-chromosomal lncRNA TTTY15 was upregulated in most PCa tissues and could promote PCa progression by sponging let-7. Patient summary We found that TTTY15 levels were frequently elevated in prostate cancer (PCa) tissues compared with those in paracancerous normal tissues in a large group of PCa patients, and we observed a tumour suppressive effect after TTTY15 knockout using CRISPR/Cas9. These results may have therapeutic implications for PCa patients.
