The lack of suitable models currently hampers our understanding of how the tumor microenvironment responds to immunotherapy treatment. Here, we present a protocol for ex vivo culture of ...patient-derived tumor fragments (PDTFs). We describe the steps for tumor collection, generation and cryopreservation of PDTFs, and their subsequent thawing. We detail culture of PDTFs and their preparation for analysis. This protocol preserves the tumor microenvironment’s composition, architecture, and cellular interactions, which can be perturbed by ex vivo treatment.
For complete details on the use and execution of this protocol, please refer to Voabil et al. (2021).1
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•Short-term ex vivo culture of patient-derived tumor tissue•Preservation of cancer, immune, and stromal cell compartments•Modeling of early immune responses to therapy•Potential for fundamental research and translational applications
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The lack of suitable models currently hampers our understanding of how the tumor microenvironment responds to immunotherapy treatment. Here, we present a protocol for ex vivo culture of patient-derived tumor fragments (PDTFs). We describe the steps for tumor collection, generation and cryopreservation of PDTFs, and their subsequent thawing. We detail culture of PDTFs and their preparation for analysis. This protocol preserves the tumor microenvironment’s composition, architecture, and cellular interactions, which can be perturbed by ex vivo treatment.
Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that ...have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase's. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.
Evidence from mouse chronic viral infection models suggests that CD8
T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential ...for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8
T lymphocyte populations with high (PD-1
), intermediate (PD-1
) and no PD-1 expression (PD-1
) from non-small-cell lung cancer patients. PD-1
T cells showed a markedly different transcriptional and metabolic profile from PD-1
and PD-1
lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1
lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1
cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.
Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative ...functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.
The overexpression of sialic acids on glycans, called hypersialylation, is a common alteration found in cancer cells. Sialylated glycans can enhance immune evasion by interacting with sialic ...acid-binding immunoglobulin-like lectin (Siglec) receptors on tumor-infiltrating immune cells. Here, we investigated the effect of sialylated glycans and their interaction with Siglec receptors on myeloid-derived suppressor cells (MDSCs). We found that MDSCs derived from the blood of lung cancer patients and tumor-bearing mice strongly express inhibitory Siglec receptors and are highly sialylated. In murine cancer models of emergency myelopoiesis, Siglec-E knockout in myeloid cells resulted in prolonged survival and increased tumor infiltration of activated T cells. Targeting suppressive myeloid cells by blocking Siglec receptors or desialylation strongly reduced their suppressive potential. We further identified CCL2 as a mediator involved in T-cell suppression upon interaction between sialoglycans and Siglec receptors on MDSCs. Our results demonstrated that sialylated glycans inhibit anticancer immunity by modulating CCL2 expression.
T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the ...dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1
hi
TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1
hi
TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1
int
and PD-1
neg
T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1
hi
expressing cells; in contrast, patients with abundance of PD-1
hi
expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1
hi
T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.
Um die Relevanz von Fahrbüchereien in der Zukunft aufzuzeigen, werden zwei Befragungen ausgewertet. Zum einen die von Studierenden der Hochschule für Angewandte Wissenschaften (HAW) Hamburg ...durchgeführte Nutzerbefragung in den Fahrbüchereien in Schleswig-Holstein in 2013 und zum anderen die bundesweit durchgeführte „Fahrbibliotheksumfrage 2012“. Beide Studien zeigen die Relevanz von Fahrbüchereien in Deutschland, jedoch bedeutet dies nicht automatisch einen Erhalt aller Fahrbüchereien. Ein Beispiel, wie dies trotzdem gelingen kann, wird anhand der Fahrbücherei der Stadtbibliothek Flensburg aufgezeigt.
Um die Relevanz von Fahrbüchereien in der Zukunft aufzuzeigen, werden zwei Befragungen ausgewertet. Zum einen die von Studierenden der Hochschule für Angewandte Wissenschaften (HAW) Hamburg ...durchgeführte Nutzerbefragung in den Fahrbüchereien in Schleswig-Holstein in 2013 und zum anderen die bundesweit durchgeführte „Fahrbibliotheksumfrage 2012“. Beide Studien zeigen die Relevanz von Fahrbüchereien in Deutschland, jedoch bedeutet dies nicht automatisch einen Erhalt aller Fahrbüchereien. Ein Beispiel, wie dies trotzdem gelingen kann, wird anhand der Fahrbücherei der Stadtbibliothek Flensburg aufgezeigt.
To demonstrate the future relevance of bookmobiles, two surveys are analyzed, one conducted by students of the Hamburg University of Applied Sciences (HAW) who interviewed users of bookmobiles in Schleswig-Holstein in 2013. The other is the “Bookmobile Survey 2012” which was executed nationwide. Both studies show the relevance of bookmobiles in Germany but do not automatically mean that they are all kept running. How this can be achieved nevertheless is shown by the example of the bookmobile of the public library of Flensburg.
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