Stromal Immune Cells Research Articles

Drug resistance in multiple myeloma: Soldiers and weapons in the bone marrow niche.

Multiple myeloma (MM) is still an incurable disease, despite considerable improvements in treatment strategies, as resistance to most currently available agents is not uncommon. In this study, data on drug resistance in MM were analyzed and led to the following conclusions: resistance occurs via intrinsic and extrinsic mechanisms, including intraclonal heterogeneity, drug efflux pumps, alterations of drug targets, the inhibition of apoptosis, increased DNA repair and interactions with the bone marrow (BM) microenvironment, cell adhesion, and the release of soluble factors. Since MM involves the BM, interactions in the MM-BM microenvironment were examined as well, with a focus on the cross-talk between BM stromal cells (BMSCs), adipocytes, osteoclasts, osteoblasts, endothelial cells, and immune cells. Given the complex mechanisms that drive MM, next-generation treatment strategies that avoid drug resistance must target both the neoplastic clone and its non-malignant environment. Possible approaches based on recent evidence include: (i) proteasome and histone deacetylases inhibitors that not only target MM but also act on BMSCs and osteoclasts; (ii) novel peptide drug conjugates that target both the MM malignant clone and angiogenesis to unleash an effective anti-MM immune response. Finally, the role of cancer stem cells in MM is unknown but given their roles in the development of solid and hematological malignancies, cancer relapse, and drug resistance, their identification and description are of paramount importance for MM management.

TMExplorer: A tumour microenvironment single-cell RNAseq database and search tool.

MotivationThe tumour microenvironment (TME) contains various cells including stromal fibroblasts, immune and malignant cells, and its composition can be elucidated using single-cell RNA sequencing (scRNA-seq). scRNA-seq datasets from several cancer types are available, yet we lack a comprehensive database to collect and present related TME data in an easily accessible format.ResultsWe therefore built a TME scRNA-seq database, and created the R package TMExplorer to facilitate investigation of the TME. TMExplorer provides an interface to easily access all available datasets and their metadata. The users can search for datasets using a thorough range of characteristics. The TMExplorer allows for examination of the TME using scRNA-seq in a way that is streamlined and allows for easy integration into already existing scRNA-seq analysis pipelines.

Immune features of the peritumoral stroma in pancreatic ductal adenocarcinoma.

BackgroundThe peritumoral stroma is a hallmark of pancreatic ductal adenocarcinoma (PDA) with implications for disease development, progression and therapy resistance. We systematically investigated immune features of the stroma in PDA patients to identify markers of clinical importance and potential therapeutic targets.MethodsTissue and blood samples of 51 PDA patients with clinical and follow-up information were included. Laser Capture Microdissection allowed us to analyze the stromal compartment in particular. Systematic immunohistochemistry, followed by software-based image analysis were conducted. Also, multiplex cytokine analyses (including 50 immune-related molecules) were performed. Functional analyses were performed using patient-derived 3D bioprints. Clinical information was used for survival analyses. Intercompartmental IL9 and IL18 gradients were assessed in matched samples of tumor epithelium, stroma, and serum of patients. Serum levels were compared to an age-matched healthy control group.ResultsStromal IL9 and IL18 are significantly associated with patient survival. While IL9 is a prognostic favorable marker (p=0.041), IL18 associates with poor patient outcomes (p=0.030). IL9 correlates with an anti-tumoral cytokine network which connects regulation of T helper (Th) 9, Th1 and Th17 cells (all: p<0.05 and r>0.5). IL18 correlates with a Th1-type cytokine phenotype and stromal CXCL12 expression (all: p<0.05 and r>0.5). Further, IL18 associates with a higher level of exhausted T cells. Inhibition of IL18 results in diminished Th1- and Th2-type cytokines. Patients with high stromal IL9 expression have a tumor-to-stroma IL9 gradient directed towards the stroma (p=0.019). Low IL18 expression associates with a tumor-to-stroma IL18 gradient away from the stroma (p=0.007). PDA patients showed higher serum levels of IL9 than healthy controls while serum IL18 levels were significantly lower than in healthy individuals. The stromal immune cell composition is distinct from the tumor epithelium. Stromal density of FoxP3+ regulatory T cells showed a tendency towards improved patient survival (p=0.071).ConclusionAn unexpected high expression of the cytokines IL9 and IL18 at different ends is of significance in the stroma of PDA and relates to opposing patient outcomes. Sub-compartmental cytokine analyses highlight the importance of a differentiated gradient assessment. The findings suggest stromal IL9 and/or IL18 as markers for patient stratification and as potential therapeutic targets. Future steps include investigating e. g. the role of local microbiota as both cytokines are also regulated by microbial compositions.

Selective poly adenylation predicts the efficacy of immunotherapy in patients with lung adenocarcinoma by multiple omics research.

The aim of this study was to find the application value of selective polyadenylation in immune cell infiltration, biological transcription function and risk assessment of survival and prognosis in lung adenocarcinoma (LUAD). The processed original mRNA expression data of LUAD were downloaded, and the expression profiles of 594 patient samples were collected. The (APA) events in TCGA-NA-SEQ data were evaluated by polyadenylation site use Index (PDUI) values, and the invasion of stromal cells and immune cells and tumor purity were calculated to group and select the differential genes. Lasso regression and stratified analysis were used to examine the role of risk scores in predicting patient outcomes. The study also used the GDSC database to predict the chemotherapeutic sensitivity of each tumor sample and used a regression method to obtain an IC50 estimate for each specific chemotherapeutic drug treatment. Then CIBERSORT algorithm was used to conduct Spearman correlation analysis, immune regulatory factor analysis and TIDE immune system function analysis for gene expression level and immune cell content. Finally, the Kaplan–Meier curve was used to analyze the correlation between stromal score and the immune score of LUAD. In this study, APA’s LUAD risk score prognostic model was constructed. KM survival analysis showed that immune score affected the prognosis of LUAD patients (P = 0.027) but the matrix score was not statistically significant (P = 0.1). We extracted 108 genes with APA events from 827 different genes and based on PUDI clustering and heat map, the survival rate of patients in the four groups was significantly different (P = 0.05). Multiple omics studies showed that risk score was significantly positively correlated with Macrophages M0, T cells Follicular helper, B cells naive and NK cells resting. It is significantly negatively correlated with dendritic cells resting, mast cells resting, monocyte, T cells CD4 memory resting and B cells memory. We further explored the relationship between the expression of immunosuppressor genes and risk score and found that ADORA2A, BTLA, CD160, CD244, CD274, CD96, CSF1R and CTLA4 genes were highly correlated with the risk score. Selective poly adenylation plays an important role in the development and progression of LUAD, immune invasion, tumor cell invasion and metastasis and biological transcription, and affects the survival and prognosis of LUAD patients.

Pan-cancer analysis combined with experimental validation revealed IL4I1 as an immunological and prognostic biomarker

Interleukin-4-induced gene 1 (IL4I1) is a secreted l-phenylalanine oxidase that deaminates phenylalanine to phenylpyruvate, releasing H2O2 and NH3 in the process. IL4I1 is mainly secreted by inflammatory antigen presenting cells and is involved in the regulation of adaptive immune responses. Furthermore, it has been reported that IL4I1 is overexpressed in a variety of tumor tissues and affects tumor development. We explored the expression patterns, correlation with clinical traits and prognostic value of IL4I1 using public databases and microarray data from sample banks. Subsequently, we used the downloaded data to score tumor stromal cells and immune cell infiltration and analyzed the correlation between IL4I1 and immune cells or immune-related molecules in combination with TIMER2.0 and GEPIA databases. The analysis showed that IL4I1 was associated with the infiltration status of various immune cells. Finally, stable IL4I1-overexpressing hepatocellular carcinoma (HCC) cell lines were established to investigate the effect of IL4I1 on cell proliferation and motor capacity. All of these results suggest that IL4I1 is a potential biomarker and therapeutic target.

Tumor-associated microglia and macrophages in glioblastoma: From basic insights to therapeutic opportunities.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Currently, the standard treatment of glioblastoma includes surgery, radiotherapy, and chemotherapy. Despite aggressive treatment, the median survival is only 15 months. GBM progression and therapeutic resistance are the results of the complex interactions between tumor cells and tumor microenvironment (TME). TME consists of several different cell types, such as stromal cells, endothelial cells and immune cells. Although GBM has the immunologically “cold” characteristic with very little lymphocyte infiltration, the TME of GBM can contain more than 30% of tumor-associated microglia and macrophages (TAMs). TAMs can release cytokines and growth factors to promote tumor proliferation, survival and metastasis progression as well as inhibit the function of immune cells. Thus, TAMs are logical therapeutic targets for GBM. In this review, we discussed the characteristics and functions of the TAMs and evaluated the state of the art of TAMs-targeting strategies in GBM. This review helps to understand how TAMs promote GBM progression and summarizes the present therapeutic interventions to target TAMs. It will possibly pave the way for new immune therapeutic avenues for GBM patients.

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.

S138: MICROENVIRONMENT-REGULATED TRANSCRIPTIONS FACTORS AHR AND HIF-1Α EXPRESSION IN TREGS PROMOTE CLL PROGRESSION BY IMPAIRING CD8+ T-CELL MEDIATED ANTI-TUMOR IMMUNITY

Background: Chronic Lymphocytic Leukemia (CLL) progression is highly dependent on complex interactions between tumor cells and the tumor microenvironment (TME). Indeed, CLL cells can modify stromal cells and immune cells to promote the survival of the leukemic clone and to escape from the immune system surveillance. Within the TME, regulatory T cells (Tregs) represent a subtype of CD4+ T cells with immunosuppressive abilities, causing the evasion of cancer cells from the immune system. We previously characterized extensively the immune microenvironment of pre-clinical CLL mouse models using mass cytometry, and we described a significant increase in the Tregs subsets with an enhanced immunosuppressive and activated phenotype compared to non-leukemic animals (Wierz et al., Blood, 2018). Interestingly, TIGIT+ Tregs are more immunosuppressive than their TIGIT- Treg counterparts and express higher levels of several transcription factors, including Ahr and Hif1α (Joller et al., Immunity, 2014), both involved in the cellular response to microenvironment-mediated stimuli. Aims: The aim of the present study is to investigate the role of AHR and HIF-1α in the suppressive ability of Tregs during CLL development. Methods: We generated conditional knock out mice (cKO) lacking Ahr or Hif1a genes exclusively in Tregs (Foxp3YFP-CreAhrfx/fx and Foxp3YFP-CreHif1afx/fx mice). We then performed adoptive transfer (AT) of CLL cells obtained from diseased Eµ-TCL1 mice into cKO and control mice. In order to decipher the mechanism by which AHR and HIF-1α pathways in Tregs affect CLL progression, we analyzed the splenic TME of recipient mice and evaluated immune checkpoint expression and cytokine production. Finally, we evaluated the suppression ability of the regulatory T cells with ex vivo suppression assays. Results: Generated cKO mice showed no sign of abnormalities or autoimmune phenotypes, and immune cells phenotyping revealed no major differences. However, we showed that CLL growth in cKO mice was drastically delayed compared to the control mice (A). Interestingly, this decrease was mitigated when CD8+ T cells were depleted. The analysis of the splenic TME in recipient cKO mice revealed an increase in IL-17 and TNF-α production, two major T-cell cytokines, in CD4+ T cells as compared to their WT counterparts. We also measured the expression of immune checkpoints and activation markers in the different T cell subpopulations and observed that Tregs lacking AHR or HIF-1α show decreased levels of the immune checkpoints CTLA-4 and TIGIT, two key proteins for the suppressive functions of Tregs. Ex vivo suppression assays demonstrated an increased proliferation of CD8+ T cells in the presence cKO Tregs (B-C). This result confirmed the decreased suppressive ability of Tregs in absence of the two transcription factors, explaining the observed delay in CLL progression in cKO mice. Image:Summary/Conclusion: Altogether, these results indicate that the TME-regulated transcription factors AHR and HIF-1α in Tregs are crucial for CLL development by promoting escape to anti-tumor immune response, and therefore represent potential therapeutic targets during CLL progression.

The Effect of Hypoxia-Induced Exosomes on Anti-Tumor Immunity and Its Implication for Immunotherapy

Hypoxia is a critical feature of solid tumors and is considered to be a key factor in promoting tumorigenesis and progression. Beyond inducing metabolic reprogramming of tumor cells to adapt to the hypoxia tumor microenvironment (TME), hypoxia can also promote tumor growth by affecting the secretion of exosomes. Exosomes are nano-sized (30-150 nm in diameter) extracellular vesicles that can carry numerous substances including lipids, proteins, nucleic acids, and metabolites. Notably, hypoxia-induced exosomes alterations not only exist in tumor cells, but also in various TME cells including stromal cells and immune cells. Besides promoting tumor invasion, angiogenesis, and drug resistance, the secretion of these altered exosomes has recently been found to negatively regulate anti-tumor immune responses. In this review, we focus on the hypoxia-induced changes in exosome secretion and found it can contributes to immune evasion and cancer progression by recruiting protumor immune cells into TME, as well as inhibiting antitumor immune cells. Next, we also describe the recent advances of exosomes in immunotherapy and future direction. In conclusion, ongoing discoveries in this field have brought new insights into hypoxia exosome-led immunosuppression, enabling the development of exosome-based therapeutics and elucidating their potential in immunotherapy.

Abstract 2542: Unraveling heterogeneity of tumor microenvironment of TNBC patients by meta-analysis of 6 breast cancer scRNAseq studies

Abstract Breast cancer can be classified into several types. Among them, triple-negative breast cancer (TNBC) is breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Since targeted therapy for breast cancer relies on the three receptors, treatment outcomes have been worst in TNBCs. However, clinical benefit of immune-checkpoint inhibitors (ICIs) with chemotherapy over chemotherapy alone was demonstrated in the KEYNOTE-355 and IMPASSION-130 trials in terms of overall survival, opening new avenue for patients with metastatic PD-L1+ TNBC. After several studies with ICIs, researchers found that the tumor microenvironment affects the response rate of immunotherapies. Therefore, to unravel the heterogeneity of tumor microenvironment of TNBC patients, we collected 28 TNBC samples from 6 public single cell RNA sequencing breast cancer studies. Results: From 124,301 cells populating tumors and tumor-microenvironment (TMEs), we first identified epithelial cells separately. Using epithelial cells alone, we identified the Baylor-proposed TNBC molecular subtype of each patient. This approach with epithelial cells revealed that 3 basal-like immune-activated (BLIA), 11 basal-like immune-suppressed (BLIS), 5 luminal- androgen receptor (LAR), and 9 unclassified subtypes. By comparing cell number and cell-cell interactions, the heterogeneity of TME-consisting cell populations between the TNBC subtypes was analyzed. Analysis of tumor-infiltrating lymphocytes revealed their difference in activation, expansion, and exhaustion programs across patients. Among the subtypes, the LAR subtype had less amount of exhausted CD8+ T cells and regulatory T cells. This finding was consistent with cell-cell interaction analysis. In the case of the LAR subtype, the interaction between regulatory T cells and CD8 T cells was less active than other subtypes. Also, the interaction between cytotoxic cells (T cells and NK cells) and regulatory T cells was less active in the LAR subtype. Additionally, tumor cells in the LAR subtype seem to inhibit cytotoxic cells to a lesser extent. When we repeated our analyses using whole cells including epithelial and immune-stromal cells, the molecular subtypes of each subject were changed (7 of 19, 36.8%). 4 of 11 BLIS were changed to BLIA, 1 of 11 BLIS were changed to mesenchymal (MES), and 2 of 5 LAR were changed to MES. Characteristics of immune cells in the epithelial-based LAR subtype were conserved in the whole cells-based MES. Conclusions:This extensive meta-analysis of public single cell RNA sequencing analysis enhances our knowledge about the heterogeneity and dynamics of the tumor microenvironment. These results offer insights about potential diagnostic and therapeutic targets for TNBC depending on their subtypes. Citation Format: Kyungsoo Kim, Sung Gwe Ahn, Soong June Bae, Chihhao Chu, Jung Hwan Ji, Jee Hung Kim, Joon Jeong. Unraveling heterogeneity of tumor microenvironment of TNBC patients by meta-analysis of 6 breast cancer scRNAseq studies [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 2542.

Abstract 1863: Mouse strain variations in drug-induced lung stromal responses

Abstract Chemotherapy has significantly improved the survival of cancer patients. However, the vast variation in treatment responses remains a major issue in the clinic. It is imperative to understand the molecular mechanisms underlying treatment failure in specific populations of patients. Genetic diversity has been recognized to mainly account for the differences in drug responses among patients. Yet, it is not feasible to map the responsible genetic variants within millions of single-nucleotide polymorphisms in the human genome in clinical settings with a limited number of subjects and insufficient genomic information. Genetically diverse mice are an ideal tool to overcome the limitations in clinical studies and model drug responses in hosts that require genetic susceptibility and resistance factors. Recent studies have also implicated a critical role for host cells (i.e., the tissue microenvironment) in building a protective “niche” for tumor cells enabling their escape from chemotherapeutic treatments. Notably, the host regenerative response upon chemotherapy “injury,” which is regarded as an intrinsic host mechanism to repair damaged tissues, may be exploited by tumor cells for their local recurrence or distant metastases. In this study, using a lung injury model, we determined how drug-induced lung stromal wound healing responses differed among eight inbred mouse strains (BALB/cJ, NSG, C57BL/6J, CH3/HeJ, CBA/J, SJL/J, A/J, and NOD/ShiLtJ). Upon either cisplatin or doxorubicin stimulation, the wound healing-associated genes Il-6, Spp1, Cxcl1, and Ccl2 were all found upregulated in lung stromal cells but showed a great variation across different strains. Furthermore, we performed single-cell RNA sequencing on the lung stromal cells and immune cells isolated from three selected mouse strains (BALB/cJ, C57BL/6J, and NOD/ShiLtJ) without and with doxorubicin treatment in vivo. The transcriptomic analyses revealed strain-specific and non-specific therapy-elicited gene signatures in different types of lung tissue cells. Collectively, our results signify an association between genetic backgrounds and drug-induced host responses in mice. The next steps of the study aim to understand how genetic diversity would functionally impact drug-induced lung tissue regeneration, lung inflammation, and post-therapy tumor relapse or metastasis in the lung. Citation Format: Carlos M. Leon, Zheng Gong, Qing Li, Jiayuan Shi, Wulin Zuo, Muneer G. Hasham, Lenny Shultz, Sheng Li, Guangwen Ren. Mouse strain variations in drug-induced lung stromal responses [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 1863.

Abstract 5211: Therapeutic resistance to anti-AR signal pathway therapies is related to compensatory pathway activation and immune suppression in mouse Pten/Trp53-deficient CRPC

Abstract Prostate cancers depend on androgens and the androgen receptor (AR) to drive androgen signaling pathways. Androgen-deprivation therapy (ADT) and second-generation antiandrogens have become the new standard of care for men with prostate cancer. However, resistance to these therapies remains a problem. TP53 mutations and PTEN loss have been associated with patients who fail to respond to anti-androgen therapies and complex interactions between AR signaling and the immune system means that these therapies have the potential to affect antitumor immune responses. Here, we characterize treatment responses of ADT alone and in combination with antiandrogen therapy using apalutamide (Apa) in a mouse model of Pten/Trp53-deficeint castrate-resistant prostate cancer (CRPC). Twenty-eight-week-old conditional Pten/Trp53-double knockout mice were randomized as untreated control or treated with ADT via orchidectomy for four weeks followed by vehicle or Apa therapy for an additional four weeks. While both treatments reduced tumor burden, there was no difference between treatments. Treatment responses in were further evaluated and were judged as favorable or non-favorable, relative to median tumor burden. Analysis of individual treatment responses indicated that 31.3% (5/16) of mice treated with ADT+Apa had tumor burden reductions &amp;gt;20% (relative to the median) compared to 6.3% (1/16) in ADT, and in both instances, 37.5% (6/16) of mice treated with ADT or ADT+Apa had tumor burden increases &amp;gt;20%. IHC showed lower nuclear/cytoplasm expression of AR in ADT+Apa treated mice and the reduction was more pronounced in favorable responders. ADT+Apa treated mice had lower gene expression levels of Ar than ADT alone and AR target genes (Fkbp5, Tmprss2, Timp4 and Nkx3.1) were decreased in ADT+Apa favorable responders whereas these target genes as well as AR-regulating genes (Myc and Igf1r, Igf1) were higher in ADT+Apa non-favorable responders. Cancer cell proliferation did not differ between treatments, but cancer glands in ADT+Apa treated mice had significantly greater IHC expression levels of cleaved caspase 3, a marker for apoptosis, and were greatest in ADT+Apa favorable responders. Levels of p-S6 ribosomal protein, a downstream target of Akt, were higher in the cancer glands of ADT+Apa but were reduced in stromal and infiltrating immune cells. Immune profiling by gene expression and flow cytometry showed enhanced macrophage involvement and reduced MDSC/granulocyte activity in ADT+Apa treated mice. There were no differences in CD8 T cell and NK cell abundance between the two cohorts, but greater cytotoxic activity was noted in all favorable responders. This study provides preclinical data that links therapeutic resistance to anti-AR signal pathways therapies in Pten/Trp53-deficeint CRPC to compensatory pathway activation and immune suppression. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazutoshi Fujita, Eri Banno, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Therapeutic resistance to anti-AR signal pathway therapies is related to compensatory pathway activation and immune suppression in mouse Pten/Trp53-deficient CRPC [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 5211.

Abstract 2878: Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®)

Abstract Immunotherapies for solid tumors need to overcome barriers within the solid tumor microenvironment (TME), including stromal cells, cancer-associated fibroblasts, endothelial cells, pericytes, and immune cells. These can comprise a significant portion of the tumor mass in many common carcinomas and contribute to immune cell dysfunction within the tumor. Stromal cells support cancer cells through several mechanisms and can promote metastasis. Tumor vasculature supplies cancer cells with nutrients and oxygen necessary for tumor growth and dissemination. Thus, targeting these components of cancer can enhance tumor rejection directly and increase sensitization of surviving tumors to immune infiltration and chemotherapeutic treatments through weakening of the tumor architecture. While there are current antiangiogenic therapies being tested clinically, they have associated toxicities in normal angiogenic processes, limiting clinical efficacy. Tumor Endothelial Marker 8 (TEM8, encoded by the ANTXR1 gene) is a highly conserved integrin-like adhesion molecule that was discovered on the endothelium of colorectal cancer, and subsequent studies identified its expression on multiple stromal cells—endothelial cells, fibroblasts, and pericytes—in the tumor microenvironment of diverse human cancer types. Little to no expression has been demonstrated in normal human stroma, indicating that this would be a good target for immunotherapy. Using a mammalian expression system, we designed and expressed a TEM8 targeting TriKE (cam1615TEM8) consisting of a humanized anti-CD16 single domain antibody, a wild-type IL-15 moiety, and an anti-TEM8 scFv. cam1615TEM8 induces NK cell degranulation and cytokine production against TEM8+ tumor and stromal cell lines (endothelial cells and fibroblasts). Using TEM8- cancer lines and a TEM8-CRISPR knockout version of A549 lung cancer lines, we show that cam1615TEM8 only activates NK cells in the presence of tumor antigen. cam1615TEM8 also preferentially stimulates TEM8+ tumor spheroid killing. Moreover, the camelid anti-CD16 nanobody selectively enhances signaling by the IL-15 moiety to NK cells, specifically promoting NK cell survival and proliferation in vitro and in vivo. In an in vivo xenogeneic mouse model, containing human NK cells and tumor, cam1615TEM8-treated mice had enhanced NK cell tumor infiltration, significantly decreased tumor growth, and enhanced survival when compared to IL-15 treated mice. Since TEM8 is highly conserved, and the scFv within the TriKE is reactive to mouse TEM8, we were able to show reduced tumor endothelial density in the tumors of mice treated with cam1615TEM8. Given the findings of this study cam1615TEM8 could be leveraged in several clinical settings by inducing NK cell specific targeting of the tumor itself and/or the tumor endothelium and stroma. Citation Format: Martin Felices, Michael Kaminski, Peter Hinderlie, Rachel Hopps, Laura Bendzick, Melissa Geller, Jeffrey S. Miller. Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®) [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 2878.

Abstract 6169: Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions

Abstract Pancreatic stellate cells (PSCs) are the main sources of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma (PDAC), which account for at least 50% of PDAC’s total tumor volume and its dense desmoplastic stroma. In recent years, immune-stromal cell interactions have been the subject of research in a variety of diseases. Interestingly, natural killer (NK) cells are relatively understudied in PDAC and may be exploited to identify new treatment opportunities. We have explored how NK cell-PSC interactions are mediated by known NK cell ligands. Human PSC (hPSC, CRC-811) or PDAC (MIA-PaCa-2, PANC-1) cell lines were co-cultured with DiO stained NK cells (NK-92, donor NK cells) for 4 hours, then target cells were assessed for apoptosis by flow cytometry using AnnexinV and Sytox stains. We found that although NK cells lyse both PSCs and PDAC cells, there was more NK cell-mediated lysis against PSCs. Using flow cytometry, we found that PSCs express the well-known MICA/B ligand, which binds the activating NKG2D receptor on NK cells to initiate its cytotoxic activity. In co-culture, neutralizing MICA/B in vitro did not completely inhibit NK cell-mediated lysis of PSCs, suggesting other interactions need to be explored. To identify other molecular determinants, NK cell ligand and receptor expression was evaluated by quantitative PCR in human PSC, PDAC, NK, and other leukocytic (K562) cell lines. Cell surface validation of highly expressed NK cell ligands and receptors were confirmed by flow cytometry and/or Western blot. NK cell ligands that were both highly expressed and localized to the cell surface were: NKG2D ligands—MICA and ULBP2 (both activating), KIR2DS4 (CD158I) ligand—HLA-A (activating), and NKp44 ligands—NKp44L and PCNA (activating and inhibitory, respectively). Their corresponding receptors were also expressed and localized to the cell surface by NK cells. These results highlight novel NK cell-PSC interactions that may mediate immune suppression in PDAC. These findings warrant further investigation. Citation Format: Zoe X. Malchiodi, Allison Fitzgerald, Elena Galvano, Louis M. Weiner. Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions [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 6169.

The utilization of artificial intelligence to produce clinically relevant scores on PD-L1 immunostained non-small cell lung cancer biopsies.

e14576 Background: Immunotherapies targeting complex programmed death-ligand 1 (PD-L1) interactions have been groundbreaking in the treatment of non-small cell lung cancer (NSCLC), offering new strategies for patients who are likely to respond. PD-L1 binds to the checkpoint PD-1 to regulate T cells, which has an important role in boosting the immune response. Identifying patients who may benefit from PD-L1 focused care is dependent on the interpretation of the drug’s associated companion diagnostic (CDx) immunohistochemistry (IHC) assay. PD-L1 IHC assays stratify NSCLC patients using a Tumor Proportion Score (TPS), a manual scoring paradigm which must be applied by a clinical pathologist. Application of the score is complex as it requires the reader to visually exclude stromal and tumor-infiltrating immune cells across a full sample, retaining PD-L1 expression information for true tumor cells only. Methods: In this study, we utilized the PD-L1 [28-8] immunohistochemistry (IHC) assay to stain 10 NCSLC specimens. Stained slides were then imaged using the AT2 scanner (Leica Biosystems, Buffalo Grove, IL) scanner and analyzed using the Visiopharm Image Analysis platform. Results: The intricacy of TPS application is time consuming for the pathologist and introduces opportunity for inter- and intra-reader variability. Our novel image analysis approach utilizes artificial intelligence (AI) to automatically denote tumor nests and exclude tumor-infiltrating immune cells. The remaining true tumor cells may then be quantified for PD-L1 expression across the full biopsy, producing a TPS for each sample. Conclusions: This method allows for great accuracy and time-efficiency in providing a TPS than traditional methods, which may result in improved patient care.

Tumour Microenvironment-Immune Cell Interactions Influencing Breast Cancer Heterogeneity and Disease Progression.

Breast cancer is a complex, dynamic disease that acquires heterogeneity through various mechanisms, allowing cancer cells to proliferate, survive and metastasise. Heterogeneity is introduced early, through the accumulation of germline and somatic mutations which initiate cancer formation. Following initiation, heterogeneity is driven by the complex interaction between intrinsic cellular factors and the extrinsic tumour microenvironment (TME). The TME consists of tumour cells and the subsequently recruited immune cells, endothelial cells, fibroblasts, adipocytes and non-cellular components of the extracellular matrix. Current research demonstrates that stromal-immune cell interactions mediated by various TME components release environmental cues, in mechanical and chemical forms, to communicate with surrounding and distant cells. These interactions are critical in facilitating the metastatic process at both the primary and secondary site, as well as introducing greater intratumoral heterogeneity and disease complexity by exerting selective pressures on cancer cells. This can result in the adaptation of cells and a feedback loop to the cancer genome, which can promote therapeutic resistance. Thus, targeting TME and immune-stromal cell interactions has been suggested as a potential therapeutic avenue given that aspects of this process are somewhat conserved between breast cancer subtypes. This mini review will discuss emerging ideas on how the interaction of various aspects of the TME contribute to increased heterogeneity and disease progression, and the therapeutic potential of targeting the TME.

Tumor Immune Microenvironment and Response to Neoadjuvant Chemotherapy in Hormone Receptor/HER2+ Early Stage Breast Cancer

BackgroundPathologic response at the time of surgery after neoadjuvant therapy for HER2 positive early breast cancer impacts both prognosis and subsequent adjuvant therapy. Comprehensive descriptions of the tumor microenvironment (TME) in patients with HER2 positive early breast cancer is not well described. We utilized standard stromal pathologist-assessed tumor infiltrating lymphocyte (TIL) quantification, quantitative multiplex immunofluorescence, and RNA-based gene pathway signatures to assess pretreatment TME characteristics associated pathologic complete response in patients with hormone receptor positive, HER2 positive early breast cancer treated in the neoadjuvant setting. MethodsWe utilized standard stromal pathologist-assessed TIL quantification, quantitative multiplex immunofluorescence, and RNA-based gene pathway signatures to assess pretreatment TME characteristics associated pathologic complete response in 28 patients with hormone receptor positive, HER2 positive early breast cancer treated in the neoadjuvant setting. ResultsPathologist-assessed stromal TILs were significantly associated with pathologic complete response (pCR). By quantitative multiplex immunofluorescence, univariate analysis revealed significant increases in CD3+, CD3+CD8-FOXP3-, CD8+ and FOXP3+ T-cell densities as well as increased immune cell aggregates in pCR patients. In subsets of paired pre/post-treatment samples, we observed significant changes in gene expression signatures in non-pCR patients and significant decreases in CD8+ densities after treatment in pCR patients. No RNA based pathway signature was associated with pCR. ConclusionTME characterization HER2 positive breast cancer patients revealed several stromal T-cell densities and immune cell aggregates associated with pCR. These results demonstrate the feasibility of these novel methods in TME evaluation and contribute to ongoing investigations of the TME in HER2+ early breast cancer to identify robust biomarkers to best identify patients eligible for systemic de-escalation strategies.

Dual Relationship Between Stromal Cells and Immune Cells in the Tumor Microenvironment.

The tumor microenvironment (TME) plays a critical role in tumorigenesis and is comprised of different components, including tumor cells, stromal cells, and immune cells. Among them, the relationship between each mediator involved in the construction of the TME can be understood by focusing on the secreting or expressing factors from each cells. Therefore, understanding the various interactions between each cellular component of the TME is necessary for precise therapeutic approaches. In carcinoma, stromal cells are well known to influence extracellular matrix (ECM) formation and tumor progression through multiple mediators. Immune cells respond to tumor cells by causing cytotoxicity or inflammatory responses. However, they are involved in tumor escape through immunoregulatory mechanisms. In general, anti-cancer therapy has mainly been focused on cancer cells themselves or the interactions between cancer cells and specific cell components. However, cancer cells directly or indirectly influence other TME partners, and members such as stromal cells and immune cells also participate in TME organization through their mutual communication. In this review, we summarized the relationship between stromal cells and immune cells in the TME and discussed the positive and negative relationships from the point of view of tumor development for use in research applications and therapeutic strategies.

Cross-Platform in-silico Analyses Exploring Tumor Immune Microenvironment with Prognostic Value in Triple-Negative Breast Cancer.

IntroductionOnly a proportion of triple-negative breast cancer (TNBC) is immunotherapy-responsive. We hypothesized that the tumor microenvironment (TME) influences the outcomes of TNBC and investigated the relevant signaling pathways.Materials and MethodsImmune score (IS) and stromal score (SS) were calculated using the ESTIMATE and correlated with the overall survival (OS) in TNBC. RNA-seq data from 115 TNBC samples and 112 normal adjacent tissues were retrieved. Validations in the methylation levels in 10 TNBC and five non-TNBC cell lines were obtained. Cox model overall survival (OS) validated the derived transcription factor (TF) genes in cBioPortal breast cancer patients.ResultsSS-low predicts a higher OS compared with SS-high patients (P = 0.0081 IS-high/SS-low patients had better OS (P = 0.045) than IS-low/SS-high patients. More macrophages were polarized to the M2 state in patients with IS-low/SS-high patients (P < 0.001). Moreover, CIBERSORTx showed more CD8+ cytotoxic T-cells in IS-high/SS-low patients (p = 0.0286) and more resting NK cells in the IS-low/SS-high TME (P = 0.0108). KEGG pathway analysis revealed that overexpressed genes were enriched in the IL-17 and cytokine-cytokine receptor interaction pathways. The lncRNA DRAIC, a tumor suppressor, was consistently deactivated in the 10 TNBC cell lines. On the cBioPortal platform, we validated that 13% of ER-negative, HER2-unamplified BC harbored IL17RA deep deletion and 25% harbored TRAF3IP2 amplification. On cBioPortal datasets, the nine altered TF genes derived from the X2K analysis showed significantly worse relapse-free survival in 2377 patients and OS in 4819 invasive BC patients than in the unaltered cohort.ConclusionOf note, the results of this integrated in silico study can only be generalized to approximately 17% of patients with TNBC, in which infiltrating stromal cells and immune cells play a determinant prognostic role.

Desmoplastic Reaction, Immune Cell Response, and Prognosis in Colorectal Cancer.

BackgroundThe relationships between tumor stromal features (such as desmoplastic reaction, myxoid stroma, and keloid-like collagen bundles) and immune cells in the colorectal carcinoma microenvironment have not yet been fully characterized.MethodsIn 908 tumors with available tissue among 4,465 incident colorectal adenocarcinoma cases in two prospective cohort studies, we examined desmoplastic reaction, myxoid stroma, and keloid-like collagen bundles. We conducted multiplex immunofluorescence for T cells [CD3, CD4, CD8, CD45RO (PTPRC), and FOXP3] and for macrophages [CD68, CD86, IRF5, MAF, and MRC1 (CD206)]. We used the inverse probability weighting method and the 4,465 incident cancer cases to adjust for selection bias.ResultsImmature desmoplastic reaction was associated with lower densities of intraepithelial CD3+CD8+CD45RO+ cells [multivariable odds ratio (OR) for the highest (vs. lowest) density category, 0.43; 95% confidence interval (CI), 0.29–0.62; Ptrend <0.0001] and stromal M1-like macrophages [the corresponding OR, 0.44; 95% CI, 0.28–0.70; Ptrend = 0.0011]. Similar relations were observed for myxoid stroma [intraepithelial CD3+CD8+CD45RO+ cells (Ptrend <0.0001) and stromal M1-like macrophages (Ptrend = 0.0007)] and for keloid-like collagen bundles (Ptrend <0.0001 for intraepithelial CD3+CD8+CD45RO+ cells). In colorectal cancer-specific survival analyses, multivariable-adjusted hazard ratios (with 95% confidence intervals) were 0.32 (0.23–0.44; Ptrend <0.0001) for mature (vs. immature) desmoplastic reaction, 0.25 (0.16–0.39; Ptrend <0.0001) for absent (vs. marked) myxoid stroma, and 0.12 (0.05–0.28; Ptrend <0.0001) for absent (vs. marked) keloid-like collagen bundles.ConclusionsImmature desmoplastic reaction and myxoid stroma were associated with lower densities of tumor intraepithelial memory cytotoxic T cells and stromal M1-like macrophages, likely reflecting interactions between tumor, immune, and stromal cells in the colorectal tumor microenvironment.