Intratumoral Immune Cell Infiltration Research Articles

Endocannabinoid Receptor 2 Function is Associated with Tumor-Associated Macrophage Accumulation and Increases in T Cell Number to Initiate a Potent Antitumor Response in a Syngeneic Murine Model of Glioblastoma.

Introduction: Glioblastoma patients have a highly immunosuppressive tumor microenvironment and systemic immunosuppression that comprise a major barrier to immune checkpoint therapy. Based on the production of endocannabinoids by glioblastomas, we explored involvement of endocannabinoid receptor 2 (CB2R), encoded by the CNR2 gene, which is predominantly expressed by immune cells, in glioblastoma-related immunosuppression. Materials & Methods: Bioinformatics of human glioblastoma databases was used to correlate enzymes involved in the synthesis and degradation of endocannabinoids, as well as CB2Rs, with patient overall survival. Intrastriatal administration of luciferase-expressing, murine GL261 glioblastoma cells was used to establish in in vivo glioblastoma model for characterization of tumor growth and intratumoral immune cell infiltration, as well as provide immune cells for in vitro co-culture experiments. Involvement of CB2Rs was determined by treatment with CB2R agonist (GW405833) or CB2R antagonist (AM630). ELISA, FACS, and immunocytochemistry were used to determine perforin, granzyme B, and surface marker levels. Results: Bioinformatics of human glioblastoma databases showed high expression of CB2R and elevated endocannabinoid production correlated with poorer prognosis, and involved immune-associated pathways. AM630treatment of GL261 glioblastoma-bearing mice induced a potent antitumor response, with survival plateauing at 50% on Day 40, when all control mice (median survival 28 days) and mice treated with GW405833 (median survival 21 days) had died. Luciferase tumor imaging revealed accelerated tumor growth by GW405833 treatment, but stable or regressing tumors in AM630-treated mice. Notably, in spleens, AM630 treatment caused an 83% decrease in monocytes/macrophages, and 1.8- and 1.6-fold increases in CD8+ and CD4+ cells, respectively. Within tumors, there was a corresponding decrease in tumor-associated macrophages (TAMs) and increase in CD8+ T cells. In vitro, lymphocytes from AM630-treated mice showed greater cytotoxic function (increased percentage of perforin- and granzyme B-positive CD8+ T cells). Discussion: These results suggest that inhibition of CB2R enhances both immunosuppressive TAM infiltration and systemic T-cell suppression through CB2R activation, and that inhibition of CB2Rs can potently counter both the immunosuppressive tumor microenvironment, as well as systemic immunosuppression in glioblastoma.

Disulfiram/Copper Activates ER Stress to Promote Immunogenic Cell Death of Oral Squamous Cell Carcinoma.

Disulfiram/copper complex (DSF/Cu) was found to have anti-tumor effects in a range of malignancies, including oral squamous cell carcinoma (OSCC), yet its precise mechanism remains unknown. It has been shown that ER stress enhances immunogenic cell death (ICD) in tumor cells, as it can influence the anti-cancer immune system favorably. In this study, we reported that DSF/Cu exhibited a marked inhibitory effect on the growth of OSCC cells, accompanied by cell apoptosis. OSCC cells treated with DSF/Cu showed the hallmarks of immunogenic cell death (ICD), including surface expression of calreticulin (CRT), heat shock protein 70 (HSP70), high mobility-group box 1 (HMGB-1) and adenosine triphosphate (ATP), thus, eliciting the maturation and activation of dendritic cells. Furthermore, we showed DSF/Cu-induced endoplasmic reticulum (ER) stress in OSCC cells. In vivo, results demonstrate that DSF/Cu inhibits tumor growth locally and alters the intratumoral immune cell infiltration and response. In conclusion, DSF/Cu suppresses OSCC development by inducing ICD and ER stress. DSF/Cu has the potential to be a new anti-tumor immunotherapy concept because of its ability to elicit ICD.

Abstract PS03-04: Tumor immune microenvironment modulates resistance to estrogen suppression in ER+ breast cancer

Abstract Despite major advances in the treatment of estrogen receptor positive (ER+) breast cancer (BC), advanced disease continues to be the main cause of death from this disease. The role of the tumor immune microenvironment (TIME) in the progression of ER+ BC and its response to treatment is not completely understood. The aim of this study was to elucidate the role the TIME in the response of ER+ tumors to estrogen deprivation (ED). We collected samples from 215 postmenopausal patients with stage I-III ER+ BC, treated with letrozole for 2-4 weeks, to induce ED. We used AQUA on pre-treatment biopsies and on-treatment surgical biopsies to assess ER, PR, HER2, and Ki67. Then, we categorized the patients’ response to ED based on the Ki67 score in on-treatment samples: ED-sensitive (ED-S) if natural log (ln) of the Ki67 score ≤1.0 or ≤2.7% Ki67+ cells vs. ED-resistant (ED-R) if ln ≥2.0 or ≥7.4% Ki67+ cells. Firstly, we assessed TIME composition by investigating stromal tumor-infiltrating lymphocytes (sTILs) in H&E-stained FFPE of on-treatment tumor sections. ED-R tumors exhibited a significantly higher stromal TILs score (p=0.0001) relative to ED-S tumors. We next prepared tissue microarrays from 227 (on-treatment) surgical sections and subjected them to cyclic immunofluorescence (CycIF) with 38 antibodies to examine their intra-tumoral immune cell infiltration. From each tumor core, we segmented single cells and labeled them as immune, cancer, or stromal cells based on expression of a set of markers. Briefly, cells that stained strongly for CD45 (leukocyte marker), and/or CD4 (T lymphocyte marker), or CD68 (macrophage marker) were labeled immune cells. CD45/CD4/CD68-negative cells were categorized as tumor if E-cadherin and/or cytokeratin-positive, or stromal cells if -negative. Next, we assessed cell specific spatial enrichment by quantifying the expression of immune markers in the area immediately adjacent to each tumor cell. Immune-suppressive T-reg (FOXP3+) cells were enriched in the ED-S tumors (p=0.0004), as well as PD1+ (exhausted) T cells (p=0.0004), and CD68+ cells (p < 0.0001) compared to ED-R tumors. ED-R tumors exhibited higher CD20+ B cells (p < 0.0001), higher CD8+ T cells (p=0.0329), in addition to higher CD45+ cells (p < 0.0001), compared to ED-S tumors. RNA-sequencing of the same surgical samples showed a higher T cells cytolytic score in ED-R relative to ED-S (p=0.0058), suggesting enhanced CD8+ T cells activity, in addition to their higher infiltration. We are currently analyzing letrozole-induced changes in TIME composition using Geomix digital spatial profiler in paired pre- and on-treatment biopsies from ED-R and ED-S tumors and will be presented at the meeting. Consistent with the CycIF findings, GSEA of hallmark gene signatures from bulk RNA-sequencing of treated tumors revealed that immune-related gene sets, such as “IFN α response”, “IFNɣ response”, and “allograft rejection” were upregulated in ED-R vs. ED-S cancers. ED-R tumors showed enrichment of CXCL9, CXCL10, and CXCL11 chemokines and their receptor, CXCR3. Publicly available datasets of patients with ER+ breast cancers showed that higher expression CXCL9 (HR 1.36; p=0.016), CXCL10 (HR 1.71; p< 0.0001), and CXCL11 (HR 1.5; p=0.0016) are predictive of shorter relapse-free survival on antiestrogen therapy. We are currently investigating whether these chemokines play a causal role in resistance to ED, and if this is phenocopied by co-cultures of ER+ BC cells and CD8+ T-cells. Conclusions ED-resistant tumors are enriched with stromal TILs and exhibit higher immune cell intra-tumoral infiltration and CD8+T cells cytolytic activity compared to ER+ tumors sensitive to estrogen suppression. In contrast, ED-S tumors showed a more immunosuppressed milieu. The role of CXCL9, CXCL10 and CXCL11 in inducing resistance to ED warrants further investigation. Citation Format: Fabiana Napolitano, Yunguan Wang, Dhivya Sudhan, Paula Gonzalez-Ericsson, Luigi Formisano, Lei Guo, M Rosario Chica-Parrado, Chang-Ching Lin, Kyung-Min Lee, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng Fang, Lin Xu, Justin Balko, Gordon Mills, Marilyne Labrie, Ariella Hanker, Carlos Arteaga. Tumor immune microenvironment modulates resistance to estrogen suppression in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-04.

Abstract A028: Disparities in suppressive immunity in Hispanic/Latina patients with hormone receptor positive breast cancer

Abstract In the US, breast cancer is the number one cancer diagnosed, and it is responsible for the most cancer related deaths among Hispanic/Latina (H/L) women. The mean age of diagnosis of breast cancer in H/L patients is 3.7 years younger than their Non-Hispanic White (NHW) counterparts. Despite being diagnosed with breast cancer at a younger age, the H/L population has a better overall survival rate across all breast cancer subtypes. This study investigates whether the immune response mounted against tumor cells in the H/L population differs from that mounted by NHW patients. We recruited and received consent from patients diagnosed with stage I- III (early stage), or stage IV/advanced (metastatic), hormone receptor positive (HR+) breast cancer. We collected blood from 66 patients (38 H/L, 28 NHW) across all stages of disease, and tumor tissue from 14 unmatched patients (7 H/L, 7 NHW), with early stage disease, at the time of tumor resection, none of whom had received neoadjuvant chemotherapy. We utilized multiparameter flow cytometry to analyze peripheral blood mononuclear cells (PBMCs), and 10X Visium for spatial transcriptomic and proteomic evaluation of patient’s tumor tissue to characterize differences in immune composition by race. We hypothesize that evaluation of intra-tumoral immune composition will reveal fewer immune suppressor cells and increased infiltration of cytotoxic immune cells in tumors from H/L patients. We also hypothesize evaluation of immune cells in circulation will mirror findings from within tumors. Suppressor immune cells, comprised of M2-polarized macrophages, myeloid derived suppressor cells (MDSCs), and T regulatory cells (Tregs) modulate T cell proliferation and cytotoxicity to affect anti-tumor immunity. We report a significant difference in the levels of both anti-tumoral and suppressive immune cells in the periphery within the H/L patient population when comparing early vs. metastatic HR+ breast cancer. This was not observed in the NHW group and there were no differences when comparing peripheral suppressor and T cell populations in H/L vs. NHW at any stage. Comparisons of intra-tumoral immune cell infiltration and differences in transcription profiles are forthcoming. The findings from this study will be some of the first to look at the immune composition in H/L women with HR+ disease, and could provide data to support larger studies leading to a paradigm shift in how we approach clinical trial design for immune checkpoint inhibition. Citation Format: Sabrina Carrel, Michelle Li, Batul Al-Zubeidy, Dominic Zavala, Edgar Gonzales, Aaron Baugh, Sofi Castanon, Robert Hsu, Dimitrios N Sidiropoulos, Yanling Ma, Michael Press, Evanthia T Roussos Torres. Disparities in suppressive immunity in Hispanic/Latina patients with hormone receptor positive breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A028.

Abstract B041: Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma

Abstract Background. Keratin 17 (K17) is a negative prognostic and predictive biomarker, overexpressed in the biologically most aggressive forms of pancreatic ductal adenocarcinoma (PDAC). In other anatomic sites and disease processes, K17 expression impacts the inflammatory microenvironment, correlating with changes in the expression of inflammatory cytokines and the infiltration of CD8+ T cells. Thus, we hypothesized that K17 expression also impacts the immune response in PDAC. Methods. We aimed to determine the relationship between intratumoral and peritumoral immune cell infiltrates versus K17 expression in tumor cells, using brightfield multiplexed immunohistochemistry (mIHC) and our suite of deep learning tools to quantitatively evaluate the expression of four biomarkers of T-cells and macrophages. The study included a multi-institutional cohort of 235 PDAC cases from Stony Brook University Hospital (n=54), Thomas Jefferson University (n=67), Cedars Sinai Medical Center (n=7) and Know Your Tumor PanCAN-Perthera (n=107). Peritumoral immune cells were scored in zones defined as stromal tissue within ≤25 µm of the closest tumor cell. Intratumor immune cells were defined as those that physically touched or overlapped with tumor cells. Immune cell counts in peritumoral and intratumoral K17-negatvie areas of the tumor were normalized to immune cell counts in K17-positive areas. Results. The density of CD8+ T cells in K17-negative peritumoral zones was greater than in K17-positive peritumoral zones in 82.6% of PDACs. Similarly, the density of intratumoral CD8+ T cells in K17-negative areas was greater than in K17-positive areas in 93.2% of cases. Less striking inverse correlations for immune cell density relative to K17 expression were found for CD4+ peritumor lymphocytes and both intratumoral and peritumor CD16+ macrophages. By contrast, CD163+ (tumor-promoting) macrophages were more numerous in K17+ peritumoral and intratumoral zones. All differences were statistically significant and independent of tumor grade, stage, histologic subtype, and neoadjuvant treatment history (p< 0.0001). Conclusions. Correlations between K17 and inflammatory cells suggests K17 expression negatively influences the number and infiltration of T-cells and contributes to the marked immunosuppressive effect of PDAC. These findings suggest that the biologic impact of K17 goes beyond its role as a negative prognostic biomarker that impacts tumor growth, metastasis, and chemoresistance, to suppress the immune response to PDAC. Understanding how K17 influences immune cell function may inform future immunotherapeutic opportunities for PDAC treatment. Citation Format: Lyanne Oblein, Sruthi Babu, Mariana Torrente-Goncalves, Lucia Roa, Michael Horowitz, Mahmudul Hasan, Danielle Fassler, Jaymie Oentoro, Emanuel Petricoin, Lynn M. Matrisian, Natalia Marchenko, Edik Blais, Felicia Allard, Ali Alkalin, Wei Jiang, Brent Larson, Andrew Hendifar, Chao Chen, Shahira Abousamra, Dimitris Samaras, Tahsin Kurc, Joel Saltz, Luisa Escobar-Hoyos, Kenneth Shroyer. Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B041.

EXTH-22. AN IND-ENABLED STUDY USING A NOVEL CHECKPOINT BLOCKADE OF LAIR1 FOR GLIOBLASTOMA THERAPY

Abstract BACKGROUND Our research study focuses on the recently discovered immune checkpoint known as Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR1), which is found to be overexpressed on tumor-associated myeloid cells (MAMC), including Tumor-associated macrophages (TAM) and Myeloid-derived suppressive cells (MDSC), in GBM and other solid cancers. The overexpression of LAIR1 in gliomas is associated with poor survival, which has prompted investigations into the potential of LAIR1 blockade (aLAIR1) as a therapeutic strategy. In preclinical mouse models, Objective: A) To investigate the mechanism of LAIR1, a new class of immune checkpoint. B) To develop strategies for blocking LAIR1 signaling as a means of cancer therapeutics, thereby enabling novel mono and combinatorial treatments for GBM. METHODS 1) Conduct coculture experiments using human TAMs and autologous CAR T cells in a 3D microgel bio-printing system, which allows us to recreate TME using patient-derived immune cells and a heterogeneous glioma model. By monitoring this coculture system, we aim to gain insights into the inhibitory effects of TAMs on T cells mediated by LAIR1. 2) Utilize tumor-burden wild-type and LAIR1 knockout mice to compare the intratumoral immune cell infiltration between WT and KO mice. 3) Employ sc-RNA-seq to identify key molecules involved in the TAM-T cell interaction. 4) Perform pathway analysis to identify signaling pathways that are influenced by this interaction. RESULTS aLAIR1 shifted macrophage polarization from M1 to M2 and increased memory CD8+ T cells intratumorally. It reversed MDSC- or M2- macrophage-mediated T-cell suppression, resulting in enhanced antitumor response as a standalone treatment and when used in combination with CAR T cell therapy in PD-1-resistant tumor models. CONCLUSIONS LAIR1 is a newly identified innate immune checkpoint that is highly expressed on TAMC in solid tumors, contributing to tumor progression and drug resistance. Blocking the LAIR1 signaling pathway shows promise as an effective approach for cancer therapy.

A Multiomics Analysis of the Close Connection between Intratumoral Microbiota and Immune Cell Infiltration in Colorectal Cancer

The colorectal intratumoral microbiome and its association with the expression of the tumor genome and immune cell infiltration remain poorly characterized. Our study aims to investigate the relationship between intratumoral microbiota with tumor immune infiltration, patient prognosis, and potential downstream signaling pathways. We collected biopsy samples of tumor tissue and paracancerous tissue from 92 patients with colorectal cancer, and acquired microbiota profiling in these samples using 16s rRNA sequencing. Meanwhile, the immune markers including CD8, FOXP3, CD163, PD-1 and PD-L1 were stained by immunohistochemistry (IHC) to identify the immune infiltration in tumors. Furthermore, we used The Cancer Genome Atlas and The Cancer Microbiome Atlas databases to conduct multiomics analysis on tumor flora and patient survival, tumor gene expression profile and potential downstream pathways. There was a significant difference in α-diversity (p = 0.00051) and β-diversity (p = 0.004) between tumor and paracancerous tissues. The β-diversity of intratumoral bacterial differed by colorectal cancer tumor stage (early vs. late stage, p = 0.049) and location (left vs. right colon, p = 0.04). Stage-related flora cluster (Porphyromonas, Lachnoclostridium, Bacteroides, Aggregatibacter, and Hungatella) were identified and found to be associated with poor prognosis in colorectal cancer patients (HR = 1.79, p = 0.015). By IHC staining, we found that expression of PD-1 and FOXP3 was significantly reduced at low abundance of stage-related bacterial cluster (p<0.05). Among of them, Hungatella was negatively associated with CD8+T cell infiltration (p<0.05) in tumor. Besides, tumor-location related flora cluster (Bacteroides and Blautia) were identified and found to be associated with good prognosis in colorectal cancer patients (HR = 0.52, p = 0.011). Expression of CD163 was decreased at high abundance of location-related bacterial cluster (p<0.05). Among of them, Blautia was negatively correlated with tumor-associated macrophage infiltration(p<0.05). Furthermore, we found that the stage-related flora cluster was positively connected with the pathway of bile acid metabolism, whereas the location-dependent cluster was negatively correlated with this pathway. We found specific intratumoral bacterial clusters that were related to tumor stage and location, and the clusters were strongly associated with tumor immune infiltration and patient prognosis. Our findings may provide new viewpoint for future research between intratumoral microbiota, metabolism pathway and tumor microenvironment.

Intratumoral Microbiota Changes with Tumor Stage and Influences the Immune Signature of Oral Squamous Cell Carcinoma.

Characterization of the oral microbiota profile through various studies has shown an association between the microbiome and oral cancer; however, stage-specific determinants of dynamic changes in microbial communities of oral cancer remain elusive. Additionally, the influence of the intratumoral microbiota on the intratumoral immune system remains largely unexplored. Therefore, this study aims to stratify microbial abundance in the early-onset and subsequent stages of oral cancer and analyze their influence on clinical-pathological and immunological features. The microbiome composition of tissue biopsy samples was identified using 16S rRNA amplicon sequencing, while intratumoral and systemic immune profiling was done with flow cytometry and immunohistochemistry-based analysis. The bacterial composition differed significantly among precancer, early cancer, and late cancer stages with the enrichment of genera Capnocytophaga, Fusobacterium, and Treponema in the cancer group, while Streptococcus and Rothia were enriched in the precancer group. Late cancer stages were significantly associated with Capnocytophaga with high predicting accuracy, while Fusobacterium was associated with early stages of cancer. A dense intermicrobial and microbiome-immune network was observed in the precancer group. At the cellular level, intratumoral immune cell infiltration of B cells and T cells (CD4+ and CD8+) was observed with enrichment of the effector memory phenotype. Naive and effector subsets of tumor-infiltrating lymphocytes (TILs) and related gene expression were found to be distinctly associated with bacterial communities; most importantly, highly abundant bacterial genera of the tumor microenvironment were either negatively correlated or not associated with the effector lymphocytes, which led to the conclusion that the tumor microenvironment favors an immunosuppressive and nonimmunogenic microbiota. IMPORTANCE The gut microbiome has been explored extensively for its importance in the modulation of systemic inflammation and immune response; in contrast, the intratumoral microbiome is less studied for its influence on immunity in cancer. Given the established correlation between intratumoral lymphocyte infiltration and patient survival in cases of solid tumors, it was pertinent to explore the extrinsic factor influencing immune cell infiltration in the tumor. Modulation of intratumoral microbiota could have a beneficial effect on the antitumor immune response. This study stratifies the microbial profile of oral squamous cell carcinoma starting from precancer to late-stage cancer and provides evidence for their immunomodulatory role in the tumor microenvironment. Our results suggest combining microbiome study with immunological signatures of tumors for their prognostic and diagnostic application.

Tumor cell-intrinsic c-Myb upregulation stimulates antitumor immunity in a murine colorectal cancer model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. While no differences in proliferation, apoptosis, and angiogenesis of tumors were evident tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAMs). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding 2-microglobulin and IFNβ, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

Design and characterization of a tumor-retained interleukin-2 cytokine/antibody fusion protein for cancer immunotherapy

Abstract Interleukin-2 (IL-2) is a pleiotropic cytokine with clinical utility in stimulating anti-cancer immunity. However, its efficacy is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory and immunosuppressive immune cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor β (IL-2Rβ) and the common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Regulatory T (Treg) cells express high levels of IL-2Rα making them more responsive to IL-2 than natural killer (NK), naïve/resting effector T (Teff), and memory T cells. To extend the half-life and selectively bias the immunostimulatory functions of IL-2 in a unimolecular format, we engineered a single-chain fusion protein, F10 immunocytokine (F10 IC), that intramolecularly fuses IL-2 to an anti-IL-2 antibody that sterically blocks IL-2Rα binding and allosterically enhances IL-2Rβ binding. However, preclinical data suggested a narrow therapeutic index (TI) for F10 IC. To improve the TI of F10 IC we designed a format of F10 IC poised for intratumoral delivery and retention. We fused collagen binding domains (CBD) to F10 IC in various topographies (F10 IC-CBD) to exploit the abundance and exposure of collagen in the tumor microenvironment. With intratumoral delivery, F10 IC-CBD is retained in the tumor and exhibits a therapeutically favorable pharmacokinetic profile. Furthermore, F10 IC-CBD increases and prolongs intratumoral immune cell infiltration, significantly slows tumor progression, minimizes F10 IC toxicity and synergizes with anti-PD-1 blockade in syngeneic mouse tumor models. We have illustrated the potential of F10 IC-CBD as a potent tumor-agnostic cancer therapy. Supported by grants from the NIH (R01 EB029341, T32 AI007417), Melanoma Research Alliance, Willowcroft Foundation and a Sanofi iAward.

In Silico Pan-Cancer Analysis Reveals Prognostic Role of the Erythroferrone (ERFE) Gene in Human Malignancies.

The erythroferrone gene (ERFE), also termed CTRP15, belongs to the C1q tumor necrosis factor-related protein (CTRP) family. Despite multiple reports about the involvement of CTRPs in cancer, the role of ERFE in cancer progression is largely unknown. We previously found that ERFE was upregulated in erythroid progenitors in myelodysplastic syndromes and strongly predicted overall survival. To understand the potential molecular interactions and identify cues for further functional investigation and the prognostic impact of ERFE in other malignancies, we performed a pan-cancer in silico analysis utilizing the Cancer Genome Atlas datasets. Our analysis shows that the ERFE mRNA is significantly overexpressed in 22 tumors and affects the prognosis in 11 cancer types. In certain tumors such as breast cancer and adrenocortical carcinoma, ERFE overexpression has been associated with the presence of oncogenic mutations and a higher tumor mutational burden. The expression of ERFE is co-regulated with the factors and pathways involved in cancer progression and metastasis, including activated pathways of the cell cycle, extracellular matrix/tumor microenvironment, G protein-coupled receptor, NOTCH, WNT, and PI3 kinase-AKT. Moreover, ERFE expression influences intratumoral immune cell infiltration. Conclusively, ERFE is aberrantly expressed in pan-cancer and can potentially function as a prognostic biomarker based on its putative functions during tumorigenesis and tumor development.

Characteristics of Immune Cell Infiltration Landscape in Colorectal Cancer to Aid in Immunotherapy

The tumor microenvironment (TME) is a complex environment composed of a variety of stromal cells and immune cells that infiltrate the tumor space. Recent clinical work has shown intratumoral immune cell infiltration.

Abstract 2911: The CEA-targeted ISAC, BDC-2034, shows preclinical efficacy associated with innate immune activation, phagocytosis, and myeloid reprogramming

Abstract Introduction: Immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors through engagement of cell surface antigens to activate tumor-associated myeloid cells and initiate a broad innate and adaptive anti-tumor immune response. We are developing the ISAC BDC-2034 to target the tumor antigen, CEA (CEACAM5), which is expressed in many solid tumors. BDC-2034 comprises our proprietary antibody, CEA1, covalently conjugated to a potent TLR7/8 agonist via a non-cleavable linker. The strong pro-phagocytic capacity and slow internalization rate of CEA1 makes it an ideal antibody for the ISAC approach. Methods: The ability of BDC-2034 to induce the secretion of pro-inflammatory cytokines was evaluated in vitro using co-cultures of tumor cells and primary immune cells (including monocytes and dendritic cells). Anti-tumor efficacy was demonstrated in vivo with xenograft and syngeneic mouse tumor models. In parallel studies, the mechanism of BDC-2034 action was assessed by quantifying intratumoral immune infiltration, cytokine levels, and transcript profile. Results: In co-cultures of CEA-expressing tumor cells and immune cells, BDC-2034 induced the secretion of cytokines and chemokines that are essential for a broad anti-tumor immune response, including IL-12p70, CXCL10, and TNFα. These effects were accompanied by myeloid cell activation as demonstrated by elevation in costimulatory surface markers such as CD40. In vivo, BDC-2034 inhibited tumor growth in multiple xenograft and syngeneic mouse tumor models having CEA expression comparable to human cancers. Consistent with the proposed mechanism of action, BDC-2034 induced intratumoral immune cell infiltration, inflammatory cytokine secretion, and myeloid re-programming in a dose-dependent and CEA-dependent fashion. The constellation of BDC-2034 effects translated to durable therapeutic activity. Conclusions: These preclinical findings demonstrate the potential of BDC-2034 to generate anti-tumor activity in CEA-expressing cancers through direct innate immune activation and the induction of adaptive anti-tumor immunity. Citation Format: Lisa K. Blum, Cecelia I. Pearson, Laughing Bear Torrez Dulgeroff, Rishali Gadkari, Angela Luo, Andrew Luo, Jennifer E. Melrose, Jess L. Nolin, Hai Li, Arthur Lee, Matthew N. Zhou, Puneet Anand, Ganapathy Sarma, Karla A. Henning, Steven J. Chapin, Shelley E. Ackerman, Romas Kudirka, Yuyi Shen, Bruce Hug, Edith A. Perez, Marcin Kowanetz, Michael N. Alonso, Brian S. Safina, David Dornan, William G. Mallet. The CEA-targeted ISAC, BDC-2034, shows preclinical efficacy associated with innate immune activation, phagocytosis, and myeloid reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2911.

Exploring the association of intratumoral immune cell infiltrates with histopathologic grade in canine mast cell tumors

Cutaneous canine mast cell tumors (ccMCTs) vary in their biological behavior, treatment, and prognosis, based on their grade. Immune cell infiltration has been associated with prognosis and response to treatments in some human cancers, and immune-targeting therapeutics are increasingly being explored in veterinary oncology. However, currently little is known about the tumor microenvironment (TME) in ccMCTs. Therefore, the objective of this study was to determine the prevalence of T lymphocytes, T regulatory lymphocytes, PD-1+ cells and macrophages in low- and high-grade ccMCTs.Thirty low-grade and 20 high-grade formalin-fixed paraffin-embedded ccMCT samples were included. Immunohistochemistry (IHC) was performed to detect CD3, FOXP3, Iba1, and PD-1 on sequential sections. Three 400x fields with the highest numbers of CD3+ cells were identified for each tumor. The percentage of CD3+, FOXP3+, and Iba1+ cells, and the number of PD-1+ cells, was quantified in each of these three “hot-spot” fields using ImageJ software.Iba1 expression was significantly greater in high-grade compared to low-grade ccMCTs (mean = 12.5% vs. 9.6%, p = 0.043). PD-1 expression was low overall, but a significantly higher number of PD-1-expressing cells was observed in high-grade ccMCTs (median 1 vs. 0, p = 0.001). No significant difference was noted in CD3 and FOXP3 expression between ccMCT grades.Macrophages and PD-1+ cells were more frequent in high-grade, compared to low-grade ccMCTs. Further studies are needed to define the role of macrophages and rare PD-1+ cells in high-grade ccMCTs.

Antitumor activity and molecular mechanism of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase family of proteins that activate other proteins in cells. Functionally, PCSK9 binds to the receptor of low-density lipoprotein particles (LDL) to regulate cholesterol metabolism and lipoprotein homeostasis in human body. PCSK9 inhibition is a novel pharmacological strategy to control hypercholesterolemia and cardiovascular diseases. Recently accumulating evidence realizes that PCSK9 possesses other roles in cells, such as regulation of tissue inflammatory response, intratumoral immune cell infiltration, and tumor progression. This review discussed the advancement of PCSK9 research on its role and underlying mechanisms in tumor development and progression. For example, PCSK9 inhibition could attenuate progression of breast cancer, glioma, colon tumor, hepatocellular cancer, prostate cancer, and lung adenocarcinoma and promote apoptosis of glioma, prostate cancer, and hepatocellular cancer cells. PCSK9 deficiency could reduce liver metastasis of B16F1 melanoma cells by lowering the circulating cholesterol levels. PCSK9 gene knockdown substantially attenuated mouse tumor growth in vivo by activation of cytotoxic T cells, although PCSK9 knockdown had no effect on morphology and growth rate of different mouse cancer cell lines in vitro. PCSK9 inhibition thus can be used to control human cancers. Future preclinical and clinical studies are warranted to define anti-tumor activity of PCSK9 inhibition.

Lirilumab and Avelumab Enhance Anti-HPV+ Cervical Cancer Activity of Natural Killer Cells via Vav1-Dependent NF-κB Disinhibition.

BackgroundWe investigated the efficacy and mechanism of the anti-KIR immunotherapy lirilumab and anti-PD-L1 immunotherapy avelumab on natural killer (NK) cell activity against HPV+ cervical cancer.MethodsNK cell-mediated lysis of autologous biopsy-derived malignant cervical squamous cells and normal cervical squamous cells were measured by europium-release cytotoxicity assays. Cytokine and granzyme B release were measured by ELISPOT effector-cell-based assays and ELISA. Murine cervical cancer tumor models were constructed to assess implanted tumor volumes over time and intratumoral immune cell infiltration. Receptor-crosslinking and plate-immobilized antibody stimulation studies, with or without p65 and Vav1 silencing, were used to investigate NF-κB pathway disinhibition in NK cells.ResultsLirilumab and avelumab each enhanced NK cell disinhibition and NK cell-mediated lysis of autologous cervical cancer cells in vitro while reducing HPV+ tumor volumes and increasing intratumoral NK cell infiltration and cytolysis in vivo. Moreover, lirilumab and avelumab each promoted NK cell NF-κB disinhibition as well as stimulated cytokine and granzyme B expression in a NF-κB-dependent manner. Lirilumab+avelumab enhanced all aforementioned effects compared to either monotherapy. Vav1 silencing eliminated disinhibition of NF-κB signaling by lirilumab and avelumab, indicating their disinhibiting effects are Vav1-dependent.ConclusionsThis study supports a novel approach to enhancing NK cell lysis against HPV+ cervical cancer cells through combining lirilumab and avelumab.

REXO4 acts as a biomarker and promotes hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality and the most common form of liver cancer. REXO4 (RNA exonuclease 4 homolog) downregulation has previously been linked to enhanced chemosensitivity in breast cancer cells. The present study sought to comprehensively clarify the functional role of REXO4 in HCC. REXO4 expression levels in HCC tumor tissues and control tissue samples were established by analyzing data from the Gene Expression Omnibus (GEO) database. The expression of REXO4 was then knocked down in HCC cell lines to explore its functional role in these cells, while a gene set enrichment analysis (GSEA) approach was used to assess the functional regulator network associated with REXO4, and the Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm was used to determine the relationship between this gene and immune cell infiltration of tumor tissues. The relationship between REXO4 and metabolic pathway was analyzed by oil red O staining. Cell Counting Kit-8 assays, colony formation, wound-healing assay, and a nude mouse subcutaneous tumor model were used to evaluate the function of REXO4 in HCC. REXO4 was highly upregulated in HCC tumors and cell lines, and was an effective predictor of HCC patient prognosis. The results indicated that the knockdown of REXO4 inhibited the proliferation and progression of HCC in vitro and in vivo. GSEA approaches also revealed REXO4 to be associated with tumor progression. Furthermore, REXO4 was associated with the degree of increase of intratumoral immune cell infiltration in HCC tissues and cells, and this gene was also linked with altered lipid metabolism in HCC cells. In summary, these analyses revealed REXO4 to be upregulated in HCC and to be associated with poor patient prognosis. In addition, this gene was closely linked to key cancer hallmark pathways and was revealed to play an important role in the susceptibility of liver tumors to immune cell infiltration and activation. Thus, targeting REXO4 may be a promising approach to treating patients with HCC in the near future.

Comparison of metabolic and immunologic responses to transarterial chemoembolization with different chemoembolic regimens in a rabbit VX2 liver tumor model.

The goal of this study was to investigate the effects of TACE using Lipiodol, Oncozene™ drug-eluting embolics (DEEs), or LUMI™-DEEs alone, or combined with bicarbonate on the metabolic and immunological tumor microenvironment in a rabbit VX2 tumor model. VX2 liver tumor-bearing rabbits were assigned to five groups. MRI and extracellular pH (pHe) mapping using Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) were performed before and after intra-arterial therapy with conventional TACE (cTACE), DEE-TACE with Idarubicin-eluting Oncozene™-DEEs, or Doxorubicin-eluting LUMI™-DEEs, each with or without prior bicarbonate infusion, and in untreated rabbits or treated with intra-arterial bicarbonate only. Imaging results were validated with immunohistochemistry (IHC) staining of cell viability (PCNA, TUNEL) and immune response (HLA-DR, CD3). Statistical analysis was performed using Mann-Whitney U test. pHe mapping revealed that combining cTACE with prior bicarbonate infusion significantly increased tumor pHe compared to control (p = 0.0175) and cTACE alone (p = 0.0025). IHC staining revealed peritumoral accumulation of HLA-DR+ antigen-presenting cells and CD3 + T-lymphocytes in controls. cTACE-treated tumors showed reduced immune infiltration, which was restored through combination with bicarbonate. DEE-TACE with Oncozene™-DEEs induced moderate intratumoral and marked peritumoral infiltration, which was slightly reduced with bicarbonate. Addition of bicarbonate prior to LUMI™-beads enhanced peritumoral immune cell infiltration compared to LUMI™-beads alone and resulted in the strongest intratumoral immune cell infiltration across all treated groups. The choice of chemoembolic regimen for TACE strongly affects post-treatment TME pHe and the ability of immune cells to accumulate and infiltrate the tumor tissue. • Combining conventional transarterial chemotherapy with prior bicarbonate infusion increases the pHe towards a more physiological value (p = 0.0025). • Peritumoral infiltration and intratumoral accumulation patterns of antigen-presenting cells and T-lymphocytes after transarterial chemotherapy were dependent on the choice of the chemoembolic regimen. • Combination of intra-arterial treatment with Doxorubicin-eluting LUMI™-beads and bicarbonate infusion resulted in the strongest intratumoral presence of immune cells (positivity index of 0.47 for HLADR+-cells and 0.62 for CD3+-cells).

Comprehensive analysis of immune related lncRNAs in the tumor microenvironment of stage II–III colorectal cancer

Long non-coding RNAs (lncRNAs) associated with immunological function have increasingly been found to act as effective prognostic biomarkers of the overall survival (OS) of colorectal cancer (CRC) patients. We sought to identify a signature of immune-related lncRNAs that offered value as a tool for the prospective prognostic evaluation of patients with stage II-III CRC. The clinical and gene expression data of CRC patients in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases was obtained and separated into a training cohort composed of 202 samples, a test cohort of 124 samples from the GSE72970 dataset, and a validation cohort of 91 samples from the GSE143985 dataset. We firstly evaluated intratumoral immune cell infiltration by conducting a Single-sample gene set enrichment analyses (ssGSEA) analysis to separate patient tumors into those with low immune cell infiltration and those with high immune cell infiltration. We then compared lncRNA and mRNA expression profiles between these two tumor types, leading us to focus on eight lncRNAs identified within the resultant mRNA-lncRNA co-expression network. Multivariate Cox regression models were then utilized to detect an immune-associated lncRNA signature that offered value for prognostic model construction. Functional analyses revealed this lncRNA signature to be associated with key immunological pathways including the JAK-STAT signaling, T cell receptor signaling, and Rap1 signaling pathways. Together, our results suggest that our immune-related 4 lncRNA signature can reliably predict stage II-III CRC patient prognosis, thereby guiding efforts to better understand this disease and to effectively treat it.

Multi-omics analysis of the expression and prognostic value of the butyrophilins in breast cancer.

Butyrophilins (BTNs) belong to the immunoglobulin superfamily of transmembrane proteins and play a role in the regulation of lymphocyte activation, several autoimmune diseases, and the progression of human cancers. However, the associated clinicopathologic characteristics and prognostic value of BTNs in breast cancer remain unknown. This study aimed to discover potential key related BTN genes and signaling pathways in breast cancer, which could provide new insights for immune-based strategies. In the present study, the mRNA expression level and prognostic value of BTN2A1, BTN3A1, BTN3A2, BTN3A3, BTNL2, BTNL9, ERMAP, and MOG were measured. Up-regulation of these genes was significantly correlated with improved overall and relapse-free survival. We then analyzed the prognostic outcomes of breast cancer subtypes, genetic alterations, interaction networks, and the functional enrichment of eight BTN family genes. Our results showed that these eight genes played essential roles in tumor progression. Furthermore, an immune infiltration analysis indicated that most candidate BTN family members were associated with intratumoral immune cell infiltration, especially that of γδ T cells. Finally, gene set enrichment analysis for a single hub gene revealed that each BTN gene played a vital role in tumor progression through immune signaling pathways. These findings provided new insights into breast cancer pathogenesis and identified eight potential biomarkers for breast cancer.