T cell exclusion, immune privilege, and the tumor microenvironment.

In 1960, Klein and colleagues found that when mice developed primary methylcholanthrene-induced sarcomas, they also developed an acquired immune resistance mediated by lymph node cells to a secondary challenge comprised of cancer cells derived from the primary tumor. The paradoxical and critical finding of the study was that this anti-cancer immune response did not control the growth of the primary tumor, despite its ability to prevent the establishment of a secondary tumor comprised of cancer cells derived from the primary tumor. The primary tumor evaded immune control by establishing an immune suppressive tumor microenvironment (TME). Unambiguous evidence for the inability in humans of a systemic immune response to eliminate immunogenic cancer cells was provided by Boon's studies thirty years later of the antigens that elicit specific CD8+ T cell responses in melanoma patients. Cloned CD8+ T cells from a melanoma patient were used to identify the antigen expressed by that patient's cancer: MAGE-A1. The explicit demonstration of the co-existence of a progressing melanoma with melanoma-specific T cells in this patient implicitly raised the question of why the T cells did not control the growth of the cancer. Moreover, Rosenberg and colleagues reported evidence of disease recurrence in melanoma patients despite very high levels of vaccine-induced circulating T cells and no evidence of antigen loss. The discovery of melanoma-specific T cells in patients led not only to vaccine strategies to increase the frequency of cancer-specific T cells in patients, but also to a more direct means for accomplishing this goal, that of adoptively transferring large numbers of in vitro expanded tumor-infiltrating lymphocytes. This approach has shown some efficacy, but has not had the dramatic success of adoptively transferring virus-specific CD8+ T cells to immunodeficient bone marrow transplant recipients with CMV infection or EBV-associated lymphoproliferative disorders. Differences in the microenvironments of virally infected tissues and cancers must account for these distinct outcomes. The more recent strategy of enhancing the function of effector T cells by targeting CTLA-4 and PD-1, immunoregulatory receptors on T cells, has been successful in subsets of patients with melanoma, NSCLC, bladder cancer, and renal cell cancer. Nevertheless, it has become apparent that even if these T cell checkpoint antagonists overcome some of the immune suppressive effects of the TME, there must be other, more fundamental inhibitory reactions in the TME to explain why most patients, especially those with colorectal cancer (CRC), ovarian cancer, prostate cancer, and pancreatic ductal adenocarcinoma (PDAC) do not exhibit objective responses to these therapies. A clue to the nature of this dominant immune suppression mediated by the TME comes from studies that have examined the spatial relationship of CD8+ effector T cells to cancer cells in three of the tumors that do not respond to anti-PD-1/anti-PD-L1: CRC, ovarian cancer and PDA. In each of these cancers, CD8+ T cells are excluded from cancer cell nests, and correlates with a poor long term clinical outcome. Thus, the TME can limit the capacity of T cells to accumulate amongst cancer cells, and one must conclude that until this problem is overcome, the full potential of other approaches to T cell-mediated tumor immunotherapy will not be realized. Recent studies have begun to explain how this form of immune suppression is mediated. Pre-clinical studies in mouse models of cancer now implicate the major stromal cell types of the TME, cancer-associated fibroblasts (CAFs) and myelomonocytic cells as being responsible for restricting the accumulation of T cells in the vicinity of cancer cells. As would be predicted, overcoming this restriction has revealed the anti-tumor effects of T cell checkpoint antagonists that had been ineffective when administered as monotherapy. I will discuss my group's recent studies of how the CAF mediates the exclusion of T cells in an autochthonous moue model of PDAC. The discovery the mechanism of exclusion has led to a potential therapeutic approach that revealed the existence of a previously unsuspected spontaneous anti-cancer immune response in these mice, and uncovered the efficacy of the anti-PD-L1 T cell checkpoint antagonist. Citation Format: Douglas T. Fearon. Immune suppression by T cell exclusion in pancreatic cancer. [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 SY41-04. doi:10.1158/1538-7445.AM2015-SY41-04

Immune checkpoint inhibition promotes T cell-mediated killing of cancer cells and can induce striking responses, but objective control of tumor growth is observed in only 10-30% of patients with cancer types that generally respond to this treatment (Fearon 2014, Cancer Immunol Res 2:187). A possible cause for this limitation of checkpoint inhibition may be an immune-privileged tumor microenvironment (TME) which excludes the cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 has recently been described as an important T cell exclusion factor in the TME-driven immune suppression. In this study we aimed to investigate whether CXCL12 inhibition by the clinical stage L-aptamer (Spiegelmer®) NOX-A12 (olaptesed pegol) is able to enhance T cell infiltration in 3D tumor-stroma spheroids, thereby facilitating effective immunotherapy. We established 3D multicellular microtissues that mimic a solid tumor with a CXCL12-abundant TME. For this purpose, CXCL12-expressing murine stromal MS-5 cells were co-cultured with solid human cancer cell lines in ultra-low attachment plates for three days. Primary human T cells isolated from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were washed and dissociated for T cell quantification by flow cytometry. T cell localization in the 3D microtissues was assessed by immunohistochemistry (IHC). In order to examine T cell activation in the spheroids, a bioluminescent reporter-based PD-1/PD-L1 blockade bioassay (Promega) was adapted to the 3D format: Jurkat-PD-1/Luc T cells were incubated with anti-PD-1 and added to NOX-A12-treated spheroids (CHO-PD-L1 + MS-5). We found that NOX-A12 increases the amount of T cells in tumor-stroma spheroids in a dose-dependent manner; flow cytometry analyses revealed a 2-3 fold increase in spheroid T cell infiltration at 10 nM NOX-A12 in all examined 3D co-culture types. Enhanced T cell infiltration in the presence of NOX-A12 was corroborated by IHC. In line with this, we found increased infiltration and activation of Jurkat-PD-1/luc T cells in the MS-5/CHO-PD-L1 spheroids treated with NOX-A12. Importantly, NOX-A12 synergized with anti-PD1-induced T cell activation. Taken together, in heterotypic 3D models that mimic the complexity of the TME, the CXCL12 antagonist NOX-A12 improved T cell-based tumor immunotherapy by increasing T cell infiltration. By modulating the CXCL12 gradients within the complex 3D structure, NOX-A12 appears to break the immune-privilege of the TME, thereby paving the way for T cell migration into the tumor. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T cell-based therapies in patients with solid cancer. Citation Format: Dirk Zboralski, Lisa Bauer, Dirk Eulberg, Axel Vater. CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1473.

BackgroundDespite the huge success of immunotherapies targeting T cells, a substantial proportion of patients experience resistance or relapse due to the immunosuppressive nature of tumor microenvironment. Increasing number of studies...

Innate immune cells in the tumor microenvironment.

Immune therapy of human pancreatic ductal adenocarcinoma (PDAC) with the T cell checkpoint antagonists, anti-CTLA-4 and anti-PD-L1, has been unsuccessful. To determine the reason for this, we studied the KPC mouse model of autochthonous PDAC in which cancer cells express KrasG12D and p53R172H, and have lost the wild type p53 allele (LOH). These tumors resemble human PDAC in not responding to treatment with anti-CTLA-4 or anti-PD-L1. PDAC-bearing mice have CD8+ T cells specific for antigen(s) expressed by cancer cells. Thus, the ineffectiveness of T cell checkpoint antagonist immune therapy is not explained by the absence of an anti-tumor immune response, and immune suppression in the tumor microenvironment must occur mainly by another means. We have shown that this immune suppressive process depends on the stromal cell known as the “cancer-associated fibroblast” (CAF) that is identified by the membrane protein marker, “fibroblast activation protein” (FAP). Conditionally depleting the FAP+ CAF (Kraman M, et al., Science 2010) leads to T cell-mediated slowing of PDAC growth, and co-treatment with anti-PD-L1 enhances this effect (Feig C, et al. PNAS 2013). Three observations directed attention to the chemokine, CXCL12, as the mediator of immune suppression: the FAP+ CAF is the source of CXCL12 mRNA in PDACs; CXCL12 protein selectively “coats” the cancer cells; and T cells are excluded from the CXCL12-coated cancer cell-containing regions of the tumor. We treated PDAC-bearing mice for 24 hr with AMD3100/Plerixafor, a small molecule antagonist of the CXCL12 receptor, CXCR4, alone or together with anti-PD-L1. Tumors from AMD3100-treated mice exhibit frequent T cells within cancer cell-containing regions, and tumors from dual treated mice have even more T cells. Importantly, whereas tumor volume doubles in control mice in 6 days, AMD3100 alone arrests tumor growth, and AMD3100 + anti-PD-L1 treatment decreases tumor volume by 15%. Both therapies eliminate almost all cancer cells, leaving a residual mass containing only PanIN, inflammatory cells, and desmoplastic matrix. These findings may be relevant not only to human PDAC, but also to colorectal and ovarian carcinoma, all of which also contain FAP+ CAFs and CXCL12-coated cancer cells, demonstrate exclusion of T cells from the vicinity of cancer cells, and are resistant to therapy with anti-PD-1/anti-PD-L1. Citation Format: Douglas T. Fearon. The basis of immune suppression in murine pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA21.

Platelets Promote Activation of the Complement System in Ovarian Cancer

Elucidating the interaction between cancer and non-cancer cells, such as blood vessels, immune cells, and other stromal cells, in the tumor microenvironment is imperative in understanding the mechanisms underlying cancer progression and metastasis, which is expected to lead to the development of new therapeutics. Sphingosine-1-phosphate is a bioactive lipid mediator that promotes cell survival, proliferation, migration, angiogenesis/lymphangiogenesis, and immune responsiveness, which are all factors involved in cancer progression. Sphingosine-1-phosphate is generated inside cancer cells by sphingosine kinases and then exported into the tumor microenvironment. Although sphingosine-1-phosphate is anticipated to play an important role in the tumor microenvironment and cancer progression, determining sphingosine-1-phosphate levels in the tumor microenvironment has been difficult due to a lack of established methods. We have recently developed a method to measure sphingosine-1-phosphate levels in the interstitial fluid that bathes cancer cells in the tumor microenvironment, and reported that high levels of sphingosine-1-phosphate exist in the tumor interstitial fluid. Importantly, sphingosine-1-phosphate can be secreted from cancer cells and non-cancer components such as immune cells and vascular/lymphatic endothelial cells in the tumor microenvironment. Furthermore, sphingosine-1-phosphate affects both cancer and non-cancer cells in the tumor microenvironment promoting cancer progression. Here, we review the roles of sphingosine-1-phosphate in the interaction between cancer and non-cancer cells in tumor microenvironment, and discuss future possibilities for targeted therapies against sphingosine-1-phosphate signaling for cancer patients.

Identification of Novel Tumor-Microenvironment-Regulating Factor That Facilitates Tumor Immune Infiltration in Colon Cancer

Parenchymal cells naturally interact, react and adapt with the environment including stromal components around them in order to maintain tissue architecture and function. However, studies have shown that this spontaneous interaction will become crucial in assisting cancer invasion. The purpose of the study was to analyze the pattern of collagen deposition and localization of matrix metalloproteinase 2 and matrix metalloproteinase 9 in the tumor microenvironment during breast cancer invasion. A standard transmission electron microscopy procedure was used together with the immunogold technique with a few modifications. The ultrastructure of fibroblasts in the vicinity of cancer cells was thick, elongated and spindle shaped with nuclear indentations. Desmoplasia was present near the cancer cells. Collagen fibers were still arranged parallel to the cancer cells and fibroblasts but were less dense than collagen fibers far from cancer cells and fibroblasts. Collagen fibers were less dense in the pericellular region because of proteolytic enzyme activity, which facilitates the invasion of breast cancer cells. In immunogold localization analysis, matrix metalloproteinase 9 had consistent localization throughout cancer cells, fibroblast and stroma. In matrix metalloproteinase 2 localization, gold conjugates were more heavily deposited in cancer cells and fibroblasts than in the stroma. Invasive breast carcinoma is not an independent entity, and its survival depends on the surrounding microenvironment.

Acidic pH derived from cancer cells may induce failed reprogramming of normal differentiated cells adjacent tumor cells and turn them into cancer cells

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Despite continual hints from preclinical and clinical research of its relevance, cancer immunology existed for many years at the periphery of cancer therapeutics. It is now the focus of intense and widespread interest after observations that blocking the activity of inhibitory receptors on T cells, known as T cell checkpoints, elicits durable clinical responses in many patients. The urgent challenge is now to understand the tissue-protective cellular elements of the tumor microenvironment (TME) that explain why the majority of patients do not respond to T cell checkpoint therapy. Analysis of human cancers and mouse models has shown that this nonresponsiveness is caused by the exclusion of T cells from the vicinity of cancer cells and that cells of the TME mediate this restriction. This review examines the immunosuppressive functions of the cells of the TME and discusses the steps of the antitumor immune reaction that, if inhibited, would diminish intratumoral T cell accumulation.

Background: The immunosuppressive tumor microenvironment (TME) is a significant barrier to improving the clinical efficacy of T cell receptor (TCR) T cell therapies in solid tumors. The TME largely consists of stromal cells such as cancer associated fibroblasts (CAF) that support tumor growth and hamper immune responses by forming a physical barrier preventing T cell infiltration and exerting immunosuppressive activity. A common cell surface antigen of CAFs with limited expression in healthy tissue is fibroblast activating protein (FAP). Dual targeting T cells expressing an anti-FAP CAR together with a PRAME-specific TCR and CD8 co-receptor (CoR) were generated using non-viral gene editing. This construct enables depletion of CAFs and disruption of the tumor’s supportive stroma and immune barriers, thereby enhancing TCR-T cell infiltration and tumor eradication. Methods: Ten scFvs against FAP were engineered into 2nd gen 4-1BB CARs and retrovirally transduced into PMBCs. FAP-CAR T cells were selected based on CAR expression using flow cytometry and cytotoxicity against FAP positive target cells. T cells were non virally gene edited with selected FAP-CARs in combination with a HLA-A*02 restricted PRAME-specific TCR and a CD8 CoR. Functionality was tested under repeated antigen stimulation in 2D and 3D in vitro cancer models expressing physiological levels of FAP and PRAME. Results: Ten anti-FAP CARs bearing different scFvs were screened based on T cell surface expression and functional activity against FAP overexpressing BT-549 cancer cells. An in vitro multi-cellular model was developed consisting of GFP-labelled cancer cells expressing physiological levels of PRAME and immortalized RFP positive fibroblasts expressing physiological levels of FAP. T cells co-expressing a PRAME TCR, CD8 CoR and 4 selected FAP-CARs were generated and tested for cytotoxicity upon repeated stimulation. Two candidates enabled multiple rounds of killing. Then, a multicomponent 3D spheroid model of non-small cell lung cancer (NSCLC) was developed combining cancer cells, monocytes, CAFs and extracellular matrix, mimicking the dual action of TME, raising a physical barrier against T cell infiltration and mediating immune suppression. Constructs containing an anti-FAP-CAR demonstrated a superior infiltration, cytotoxicity and cytokine secretion profile compared to TCR-T cells without CAR armoring. Conclusion: Dual targeting T cells incorporating both a FAP-CAR and a PRAME TCR demonstrated successful infiltration and effective eradication of both cancer cells and stromal cells within our proprietary, multicomponent 3D spheroid model mimicking the harsh NSCLC TME. This approach holds promise to tackle a major challenge in solid tumor therapy, increasing T cell infiltration and eliciting potent, durable anti-tumor responses. Citation Format: Paul Najm, Kathrin Skibbe, Mikhail Steklov, Panagiota A. Sotiropoulou, Marleen M. Van Loenen. Dual targeting of cancer and stromal cells, by combining a FAP-CAR and a PRAME TCR, enhances T cell infiltration and efficient eradication of a wide range of hard-to-treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6109.

Immune checkpoint inhibitors promote T cell-mediated killing of cancer cells; however, only a subset of patients benefit from the treatment. A possible reason for this limitation may be that the tumor microenvironment (TME) is immune privileged, which may exclude cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 is key to the TME-driven immune suppression. In this study, we investigated the potential of CXCL12 inhibition by use of the clinical-stage l-RNA-aptamer NOX-A12 (olaptesed pegol) to increase the number of tumor-infiltrating lymphocytes. We used heterotypic tumor-stroma spheroids that mimic a solid tumor with a CXCL12-abundant TME. NOX-A12 enhanced the infiltration of T and NK cells in a dose-dependent manner. NOX-A12 and PD-1 checkpoint inhibition synergistically activated T cells in the spheroids, indicating that the agents complement each other. The findings were validated in vivo in a syngeneic murine model of colorectal cancer in which the addition of NOX-A12 improved anti-PD-1 therapy. Taken together, our work shows that CXCL12 inhibition can break the immune-privileged status of the TME by paving the way for immune effector cells to enter into the tumor, thereby broadening the applicability of checkpoint inhibitors in cancer patients. Cancer Immunol Res; 5(11); 950-6. ©2017 AACR.

Lactate, the main product of tumor glycolysis is considered one of the most crucial metabolites in the tumor microenvironment. The reprogrammed cancer cell metabolism is closely associated with the enhanced rate of tumor glycolysis leading to excess lactate production concomitant with increased extracellular acidification. This decline in pH in the vicinity of cancer cells is largely attributed to reprogrammed tumor glycolysis (Warburg effect). Substantial literature data to date suggest that lactate is not merely a waste product of tumor glycolysis, albeit serves as the main fuel to meet the anabolic requirements of cancer cells. Lactate plays a critical role in tumor growth, migration and invasion, tumor metastasis, tumor microenvironment and immune modulation. This review summarizes the current knowledge about the role of lactate in tumor glycolysis, its fate and transporters, lactate shuttle, and metabolic symbiosis. It also condenses the role of lactate in the tumor microenvironment and immune invasion and the development of therapeutic strategies.