Human Tumor Tissue Microarrays Research Articles

Abstract 4094: Immune modulatory cancer vaccines against IDO1 and PD-L1 trigger distinct pathways and cooperatively reduce tumor growth in preclinical models

Abstract While immune checkpoint inhibitors have shown clinical efficacy in many cancers, drug and immune resistance remain challenging. Indoleamine 2,3-dehydrogenase (IDO1) and Programmed Death Ligand 1 (PD-L1) both contribute to immunosuppression, leading to immune escape and cancer progression. In this context, vaccination to promote T-cell immunity against IDO1+ and PD-L1+ cells is an attractive strategy that demonstrated encouraging clinical results in melanoma (Kjeldsen et al. Nat Med 2021). Our study aims to further evaluate the efficacy and mode of action of combined IDO1 and PD-L1 peptide vaccines. Mice injected with tumor cells and treated with IDO1 or PD-L1 peptides were monitored for tumor growth. Vaccine activity was determined by IFNγ Elispot assay. Gene expression analysis was performed on tumors using Nanostring nCounter PanCancer IO360 Panel. IDO1 and PD-L1 expression were also examined via multiplex immunofluorescence in a human tumor tissue microarray (TMA) panel. Human IDO1 and PD-L1 specific CD4+ T cell clones were isolated and expanded from melanoma patients treated with IDO1/IO102 or PD-L1/IO103 peptide vaccines in combination with aPD1 therapy. Immunofluorescence analysis of murine tumor sections showed expression of IDO1 and PD-L1 by different cell populations. Vaccination against each target led to expansion of specific T cells in splenocytes and reduced tumor growth in MC38 and CT26 models. Combining IDO1 and PD-L1 treatments further enhanced the anti-tumor response. Gene expression analysis of tumors revealed distinct signatures following monotherapy and combination treatments. While PD-L1 vaccination predominantly enhanced the cytotoxic effector T cell function in the tumor microenvironment (TME) and concomitantly increased gene signatures associated with enhanced activation of T effector cells, IDO1 vaccine primarily exerted its effect via reduction in the immune suppression loop. TMA analysis of IDO1+ and PD-L1+ cells in human tumors also revealed that they represent largely distinct cell populations. Lastly, IDO1 and PDL1-specific CD4+ T cell clones from melanoma patients treated with IO102-IO103 vaccine could selectively target cells differentially expressing IDO1 or PDL1. Our data collectively show that cells expressing IDO1 and PD-L1 represent distinct populations in the TME thus targetable by the IDO1-PD-L1 vaccination approach. The vaccines targeting IDO1 and PD-L1 cooperatively reduce tumor outgrowth, and each contributes to the anti-tumor effect through distinct molecular programs regulating immunosuppression in the TME and T cell activation respectively. These data are supported by ex vivo functional assays using target specific T cells from vaccinated patients. Altogether, our data support the use of a dual antigen approach to reduce the immunosuppression and enhance anti-tumor effect. Citation Format: Marion Chapellier, Evelina Martinenaite, Preeyam Patel, Lea Svendsen, Shamaila Munir Ahmad, Hannah Jorinde Glöckner, Marco Carretta, Erika Sutanto-Ward, James DuHadaway, Souvik Dey, Shih-Chun Shen, Dema Ghaban, Marcos Iglesias, Megan Biller, Inés Lecoq, Alexander J. Muller, Mads Hald Andersen, Ayako Wakatsuki Pedersen. Immune modulatory cancer vaccines against IDO1 and PD-L1 trigger distinct pathways and cooperatively reduce tumor growth in preclinical models [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 4094.

Lysine acetylation of NKG2D ligand Rae-1 stabilizes the protein and sensitizes tumor cells to NKG2D immune surveillance.

Shedding, loss of expression, or internalization of natural killer group 2, member D (NKG2D) ligands from the tumor cell surface leads to immune evasion, which is associated with poor prognosis in patients with cancer. In many cancers, matrix metalloproteinases cause the proteolytic shedding of NKG2D ligands. However, it remained unclear how to protect NKG2D ligands from shedding. Here, we showed that the shedding of the mouse NKG2D ligand Rae-1 can be prevented by two critical acetyltransferases, GCN5 and PCAF, which acetylate the lysine residues of Rae-1 to avoid shedding both in vitro and in vivo. In contrast, mutations at lysines 80 and 87 of Rae-1 abrogated this acetylation and thereby desensitized tumor cells to NKG2D-dependent immune surveillance. Notably, the protein levels of GCN5 correlated with the expression levels of the human NKG2D ligand ULPB1 in a human tumor tissue microarray and, more importantly, with prolonged overall survival in many cancers. Our results suggest that the acetylation of Rae-1 protein at lysines 80 and 87 by GCN5 and PCAF protects Rae-1 from shedding so as to activate NKG2D-dependent immune surveillance. This discovery may shed light on new targets for NKG2D immunotherapy in cancer treatment.

Abstract C104: Therapeutic targeting of TREM2+ tumor-associated macrophages as a means of overcoming checkpoint inhibitor resistance

Abstract Tumor-associated macrophages (TAMs) are a major sources of innate checkpoint inhibitor (CPI) resistance, as they subvert anti-tumor immunity through immunosuppression and supporting tumor growth. In patients, high levels of TAMs predict poor prognosis across multiple solid tumor indications. Therefore, therapeutic targeting of TAMs by impacting their survival and/or modulating their suppressive function is a promising strategy to augment response rates in solid tumor indications, as well as overcome resistance to CPI therapies. We and others have identified the transmembrane protein triggering receptor expressed on myeloid cells-2 (TREM2) as a highly enriched TAM target. TREM2 is comprised of one IgV domain but lacks any known intracellular signaling motifs. However, TREM2 associates with TYRO protein tyrosine kinase-binding protein (TYROBP), which contains an intracellular immunoreceptor tyrosine-baased activation motif (ITAM), allowing for TREM2/TYROBP-mediated signaling to occur. Screening of human tumor tissue microarrays for TREM2 protein demonstrated that TREM2+ TAMs were present in multiple human solid tumors and correlated with disease severity in some indications. Furthermore, in a variety of tumor types, TREM2 mRNA expression negatively correlated with patient survival. To target TAMs, we developed an anti-TREM2 monoclonal antibody (mAb), termed PY314m, that demonstrated compelling anti-tumor activity either as single agent in CPI-sensitive syngeneic tumor models or in combination with anti-PD-1 in CPI-resistant mouse syngeneic tumor models. Mechanistically, PY314m reduced a subset of TAMs, namely the MHCIILow ArginaseHigh M2-like TAMs, expanded MHCIIHigh M1-like TAMs, and induced both innate and adaptive pro-inflammatory cytokine production. By depleting M2-like TAMs and inducing expansion of M1-like TAMs, PY314m treatment removes and modulates multiple immunosuppressive mechanisms. The combination treatment of PY314m and anti-PD-1 mAb produced long-term immunological memory as evidenced by the resistance to tumor rechallenge in mice cured of their tumors. Collectively these findings suggest that anti-TREM2 mAb therapy could be used to overcome CPI resistance in humans. To this end, we developed a humanized version of PY314m termed PY314, which was well-tolerated and showed good pharmacokinetic properties in pilot, repeat dose non-GLP studies in non-human primates. Our ongoing efforts are aimed at better defining CPI-resistant patient populations that will maximally benefit from PY314 therapy. Citation Format: Mikhail Binnewies, Marwan Abushawish, Tian Lee, Tiep Le, Joshua L. Pollack, Erick Lu, Amanda Chen, Ranna Mehta, Nadine Jahchan, Vicky Huang, Xiaoyan Du, Subhadra Dash, Manith Norng, Aritra Pal, Kevin P. Baker, Venkataraman Sriram. Therapeutic targeting of TREM2+ tumor-associated macrophages as a means of overcoming checkpoint inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C104. doi:10.1158/1535-7163.TARG-19-C104

Overexpression of the VRK1 kinase, which is associated with breast cancer, induces a mesenchymal to epithelial transition in mammary epithelial cells.

Vaccinia-related kinase 1 (VRK1) is a pro-proliferative nuclear kinase. Mice engrafted with VRK1-depleted MDA-MB-231 breast cancer cells have been shown to develop fewer distal metastases than controls, suggesting VRK1 might play a role in cell migration, invasion, and/or colonization. In work described herein, we investigated the impact of VRK1 overexpression on human mammary epithelial cells. In 2D culture, VRK1 overexpression diminishes cell migration and invasion and impairs the migration-associated processes of cell spreading and cytoskeletal rearrangement. VRK1-overexpressing cells show reduced accumulation of the mesenchymal marker vimentin and increased accumulation of the epithelial markers E-cadherin and claudin-1. VRK1 overexpression also leads to reduced levels of the transcriptional repressors snail, slug, and twist1. Cumulatively, these data indicate that VRK1 overexpression augments the epithelial properties of both MCF10a and MDA-MB-231 cells. We further studied the impact of VRK1 on the epithelial properties of MCF10a cells in 3D matrigel culture, in which cells proliferate and form epithelial sheets that mature into hollow spherical acini. VRK1 overexpression significantly accelerates the initial stages of cell proliferation, leading to larger acini that nevertheless differentiate and mature. Our analysis of human tumor tissue microarrays (TMAs) revealed that VRK1 protein levels are higher in lymph node metastases than in patient-matched mammary tumors. Using public databases, we determined that VRK1 is among the top 10% of overexpressed transcripts in multiple subtypes of invasive breast cancer, and that high levels of VRK1 expression are correlated with decreased relapse-free survival. In sum, overexpression of VRK1, by regulating the transcription repressors snail, slug, and twist1, can promote a mesenchymal-to-epithelial transition (MET) in cell culture. VRK1-mediated MET might facilitate the colonization of distal sites by metastatic breast cancer cells, providing some insight into the frequent association of VRK1 overexpression with breast malignancies and the correlation between VRK1 overexpression and poor clinical outcome.

Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment

BackgroundMyeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity.MethodsCB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively.ResultsCB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers.ConclusionsThese results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

Abstract 4904: Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology

Abstract Heat induced target retrieval (HITR) is a widely adopted approach for Immunohistochemistry (IHC) and in situ hybridization (ISH) applications on formalin fixed and paraffin embedded (FFPE) tissues to facilitate target access and increase detection sensitivity. A variety of factors, such as fixative type, fixation time, tissue type, epitope abundance, antibody affinity, and probe length greatly affect the efficiency of HITR. Given such variability, over the decades, multiple HITR buffer formulations and protocols were invented to fit the needs in different experimental scenarios. We have developed a novel HITR solution that allows highly efficient target retrieval for RNA ISH application based on the RNAscope technology. When tested on 15 different tumor types, the new HITR solution resulted in significantly better sensitivity and/or tissue morphology comparing to using traditional HITR buffers, including Citrate, EDTA, and Tris based buffers with various pH. Furthermore, this new HITR solution is highly tolerant to sample variability. When used on multiple human tumor tissue microarrays (TMAs), including breast, colon, lung, and gastric cancers, the new HITR solution improved overall signal-to-noise ratio and reduced core disqualification rate, compared to traditional citrate based HITR buffer for ISH. Moreover, when tested on multiple mouse tissues fixed with different types of fixatives for varying periods of time (6 - 72 hours), a single experimental condition with the new HITR solution showed comparable results in both detection sensitivity and morphological integrity in all the samples. It also allowed detection of a target (Tbp) with extremely low abundance (<5 copies/cell). Given that all the RNAscope probes are synthetic oligonucleotide probes with length shorter than 60 bases, the new HITR solution, in combination with the RNAscope technology, will allow universal detection of any RNA targets with single molecule detection sensitivity in FFPE tissues with minimal requirement for analytical condition optimization. Citation Format: Li-chong Wang, Liuliu Pan, Kuang-Jung Chang, Daniel Kim, Xingyong Wu, Hongwei Wang, Casey Kernag, Bingqing Zhang, Mingxiao He, Nan Su, Xiao-Jun Ma, Yuling Luo. Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2015-4904

Stimulating anti-tumor immune response: the problem of regulatory T-cells

Approaches to stimulating an anti-tumor immune response in glioblastoma patients have been a subject of interest in preclinical and clinical brain tumor research for decades. To this end, substantial effort has been directed towards inhibiting regulatory T cells (Tregs), which promote immune tolerance and limit the effectiveness of therapies that depend on evoking an anti-tumor immune response. In the current issue of Neuro-Oncology, Wainwright et al. (1) investigate the origin of glioblastoma-associated Tregs, specifically whether they are thymus derived vs. induced; this knowledge may be critically important to developing effective approaches to limiting Treg anti-tumor immunosuppression. To achieve their objective, the authors used a murine, syngeneic, immunocompetent, orthotopic engraftment model to evaluate the effect of thymectomy on systemically administered anti-Treg CD25 mAb and found that the thymus is necessary for animal subjects to experience a survival benefit from this Treg-depleting treatment. To establish the relevance of the experimental model observations to glioblastoma in patients, a human tumor tissue microarray was examined for expression of the thymus-derived marker Helios in tumor-resident Tregs; the results showed that the majority of tumor-associated T-cells were Helios positive. In total, the results of this elegant study suggest that attempts to suppress Tregs, in association with therapies for evoking patient anti-tumor immune response, need to be targeted to the periphery, where Treg development and expansion predominates. However, since brain tumor patients present with Tregs already infiltrating their malignancy, it may additionally be necessary to target Tregs in the brain. This, in turn, would require the use of Treg-suppressive therapeutics with favorable biodistribution properties. The featured article emphasizes the increasing interest in exploiting patients' immune responses to combat their disease, while informing us of the complexity of cell types and processes involved in the immune response to cancer. How best to manipulate immune function to achieve improved outcomes in brain tumor patients is certain to remain a subject of keen interest, with innovative approaches such as Treg depletion through antibody administration, providing reason for optimism about the eventual benefits of immunotherapy in neuro-oncology practice.

Loss of ANXA7 Expression is Associated with Poor Patient Survival in Ovarian Cancer

Epithelial ovarian cancer is morphologically heterogeneous being classified as serous, endometrioid, clear cell, or mucinous. Molecular genetic analysis has suggested a role for tumor suppressor genes located at chromosome 10q in epithelial ovarian cancer pathogenesis. Our objective is to evaluate the expression of ANXA7, a novel tumor suppressor gene located on 10q21, in these epithelial ovarian cancer subtypes, and to investigate its correlation with patient survival. ANXA7 is ubiquitously expressed in small amounts in nearly every normal cell and the Anxa7 (+/-) knockout mouse has a cancer prone phenotype. Altered ANXA7 protein levels are associated with prognostically challenging aggressive forms of prostate and breast cancer. So far, information is not available regarding the association of ANXA7 expression in ovarian cancer and patient survival. Therefore, we used human tumor tissue microarray (TMA) technology in order to evaluate the ANXA7 immunoreactivity as possible diagnostic and/or prognostic marker of ovarian cancer by immunoperoxidase assay using ANXA7 monoclonal antibody. Using a 129 case diagnostic human tumor tissue microarray, we report that the expression of ANXA7 is significantly reduced and is associated with disease progression. Furthermore, using a separate 301 case retrospective prognostic tumor tissue microarray, we find that loss of ANXA7 expression is also significantly associated with poor over-all patient survival. We conclude that ANXA7 may be a new prognostic marker or a target for improving the treatment efficiency of patients with ovarian cancers.

TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors

BackgroundMembers of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2).MethodsOncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target.ResultsOverexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro.ConclusionThis study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.

Heterogeneous expression of the aquaporin 1 (AQP1) water channel in tumors of the prostate, breast, ovary, colon and lung: A study using high density multiple human tumor tissue microarrays

Aquaporin 1 (AQP1) water channels are membrane proteins that control the permeability of endothelial and epithelial barriers by facilitating water movement across cell membranes. Recent studies suggest that AQP1 may be responsible for the high vascular permeability and interstitial fluid pressure in tumors of the brain, colon, breast and pancreas. AQP1 may also play a role in tumor angiogenesis and may be involved in development of effusions or edema fluid. The aim of the present study was to use immunohistochemistry and semi-quantitative histomorphometric analysis to compare the distribution and relative abundance of AQP1 on NCI TARP human multiple tumor tissue microarrays (TMAs) with normal tissues represented on the CHTN TMAs. Immunohistochemistry and semi-quantitative histomorphometric analysis were used to compare the distribution of AQP1 in tumors of the prostate, colon, lung, breast and ovary represented on TARP TMAs with their normal counterparts on CHTN TMAs. AQP1 was expressed in capillary endothelia of all normal tissues. In most tumors AQP1 was confined to endothelial barriers. AQP1 expression was marginally higher in microvascular structures in prostate and ovarian tumors and was higher in advanced mammary and colorectal carcinomas where AQP1 immunoreactivity was also seen in some neoplastic tumor cells. In conclusion, the AQP1 water channel is an excellent marker of microvasculature but it is heterogeneously expressed in different human tumors and not necessarily expressed in all neoplastic cells. Increased AQP1 expression in some human adenocarcinomas may be a consequence of angiogenesis and important for the formation or clearance of tumor edema.

ANX7 as a bio-marker in prostate and breast cancer progression.

The ANX7 gene codes for a Ca2+-activated GTPase, which has been implicated in both exocytotic secretion in cells and control of growth. In this review, we summarize information regarding increased tumor frequency in the Anx7 knockout mice, ANX7 growth suppression of human cancer cell lines, and ANX7 expression in human tumor tissue micro-arrays. The loss of ANX7 is significant in metastatic and hormone refractory prostate cancer compared to benign prostatic hyperplasia. In addition, ANX7 expression has prognostic value for predicting survival of breast cancer patients.