A comprehensive single-cell map of T cell exhaustion-associated immune environments in human breast cancer

Immune checkpoint therapy in breast cancer remains restricted to triple negative patients, and long-term clinical benefit is rare. The primary aim of immune checkpoint blockade is to prevent or reverse exhausted T cell states, but T cell exhaustion in breast tumors is not well understood. Here, we use single-cell transcriptomics combined with imaging mass cytometry to systematically study immune environments of human breast tumors that either do or do not contain exhausted T cells, with a focus on luminal subtypes. We find that the presence of a PD-1high exhaustion-like T cell phenotype is associated with an inflammatory immune environment with a characteristic cytotoxic profile, increased myeloid cell activation, evidence for elevated immunomodulatory, chemotactic, and cytokine signaling, and accumulation of natural killer T cells. Tumors harboring exhausted-like T cells show increased expression of MHC-I on tumor cells and of CXCL13 on T cells, as well as altered spatial organization with more immature rather than mature tertiary lymphoid structures. Our data reveal fundamental differences between immune environments with and without exhausted T cells within luminal breast cancer, and show that expression of PD-1 and CXCL13 on T cells, and MHC-I - but not PD-L1 - on tumor cells are strong distinguishing features between these environments

Astrocyte Depletion Impairs Redox Homeostasis and Triggers Neuronal Loss in the Adult CNS

Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood. With the use of conditional, astrocyte-restricted protein synthesis termination, we found that selective paralysis of GFAP(+) astrocytes in vivo led to rapid neuronal cell loss and severe motor deficits. This occurred while structural astroglial support still persisted and in the absence of any major microvascular damage. Whereas loss of astrocyte function did lead to microglial activation, this had no impact on the neuronal loss and clinical decline. Neuronal injury was caused by oxidative stress resulting from the reduced redox scavenging capability of dysfunctional astrocytes and could be prevented by the in vivo treatment with scavengers of reactive oxygen and nitrogen species (ROS/RNS). Our results suggest that the subpopulation of GFAP(+) astrocytes maintain neuronal health by controlling redox homeostasis in the adult CNS

Whole-Exome Sequencing Reveals High Mutational Concordance between Primary and Matched Recurrent Triple-Negative Breast Cancers

PURPOSE Triple-negative breast cancer (TNBC) is a molecularly complex and heterogeneous breast cancer subtype with distinct biological features and clinical behavior. Although TNBC is associated with an increased risk of metastasis and recurrence, the molecular mechanisms underlying TNBC metastasis remain unclear. We performed whole-exome sequencing (WES) analysis of primary TNBC and paired recurrent tumors to investigate the genetic profile of TNBC. METHODS Genomic DNA extracted from 35 formalin-fixed paraffin-embedded tissue samples from 26 TNBC patients was subjected to WES. Of these, 15 were primary tumors that did not have recurrence, and 11 were primary tumors that had recurrence (nine paired primary and recurrent tumors). Tumors were analyzed for single-nucleotide variants and insertions/deletions. RESULTS The tumor mutational burden (TMB) was 7.6 variants/megabase in primary tumors that recurred (n = 9); 8.2 variants/megabase in corresponding recurrent tumors (n = 9); and 7.3 variants/megabase in primary tumors that did not recur (n = 15). MUC3A was the most frequently mutated gene in all groups. Mutations in MAP3K1 and MUC16 were more common in our dataset. No alterations in PI3KCA were detected in our dataset. CONCLUSIONS We found similar mutational profiles between primary and paired recurrent tumors, suggesting that genomic features may be retained during local recurrence

Integrated Analysis Of Immunotherapy Treated Clear Cell Renal Cell Carcinomas: An Exploratory Study

Molecular or immunological differences between responders and nonresponders to immune checkpoint inhibitors (ICIs) of clear cell renal cell carcinomas (ccRCCs) remain incompletely understood. To address this question, we performed next-generation sequencing, methylation analysis, genome wide copy number analysis, targeted RNA sequencing and T-cell receptor sequencing, and we studied frequencies of tumor-infiltrating CD8+ T cells, presence of tertiary lymphoid structures (TLS) and PD-L1 expression in 8 treatment-naive ccRCC patients subsequently treated with ICI (3 responders, 5 nonresponders). Unexpectedly, we identified decreased frequencies of CD8+ tumor-infiltrating T cells and TLS, and a decreased expression of PD-L1 in ICI responders when compared with nonresponders. However, neither tumor-specific genetic alterations nor gene expression profiles correlated with response to ICI or the observed immune features. Our results underline the challenge to stratify ccRCC patients for immunotherapy based on routinely available pathologic primary tumor material, even with advanced technologies. Our findings emphasize the analysis of pretreated metastatic tissue in line with recent observations describing treatment effects on the tumor microenvironment. In addition, our data call for further investigation of additional parameters in a larger ccRCC cohort to understand the mechanistic implications of the observed differences in tumor-infiltrating CD8+ T cells, TLS, and PD-L1 expression

scROSHI: robust supervised hierarchical identification of single cells

Identifying cell types based on expression profiles is a pillar of single cell analysis. Existing machine-learning methods identify predictive features from annotated training data, which are often not available in early-stage studies. This can lead to overfitting and inferior performance when applied to new data. To address these challenges we present scROSHI, which utilizes previously obtained cell type-specific gene lists and does not require training or the existence of annotated data. By respecting the hierarchical nature of cell type relationships and assigning cells consecutively to more specialized identities, excellent prediction performance is achieved. In a benchmark based on publicly available PBMC data sets, scROSHI outperforms competing methods when training data are limited or the diversity between experiments is large

Differential PD-1/LAG-3 expression and immune phenotypes in metastatic sites of breast cancer

Background: A dual blockade against the novel immune checkpoint inhibitor lymphocyte activation gene-3 (LAG-3) and programmed cell death protein-1 (PD-1) is currently considered in advanced breast cancer. Nevertheless, PD-1 or LAG-3 expression within distant metastatic breast cancer tissue remains understudied. Methods: To address this knowledge gap, we investigated the PD-1 and LAG-3 expression in combination with the CD8-based immune phenotype in intrapatient matched primary tumor distant metastases, representing 95 breast cancer patients with metastases occurring at four different anatomical locations. The immune phenotype was categorized into 2 categories: inflamed corresponding to the clinical category "hot" and exhausted or desert consistent with clinically "cold" tumors. Results: Metastases of "cold" primary tumors always remained "cold" at their matched metastatic site. Expression of PD-1/LAG-3 was associated with a "hot" immune phenotype in both the primary tumors and metastases. We could not observe any association between the immune phenotype and the breast cancer molecular subtype. Brain and soft tissue metastases were more commonly inflamed with signs of exhaustion than other anatomical sites of metastases. Taken together, (i) the immune phenotype varied between sites of distant metastases, and (ii) PD-1+/LAG-3+ was strongly associated with a "hot" immune phenotype and (iii) was most prevalent in brain and soft tissue metastases among distant metastases. Conclusions: Our data strongly support an integrated analysis of the immune phenotype together with the PD-1/LAG-3 expression in distant metastases to identify patients with inflamed but exhausted tumors. This may eventually improve the stratification and likelihood for advanced breast cancer patients to profit from immunotherapy. Keywords: Breast cancer; Immune checkpoint receptors; Immune phenotype; Metastasis; Tumor immunology

Regulation of erythropoietin expression in renal cancer and primary established cell lines thereof

GesamtdissertationDie Genexpression Erythropoietins (EPO) ist durch ihre ausserordentliche Gewebe- und Zellspezifitaet gekennzeichnet. Fuer die Expression notwendig ist die Aktivierung des HIF (hypoxia-inducible factor)-Transkriptionskomplexes, der durch das pVHL (Protein von Hippel-Lindau) reguliert wird. Ungeachtet dessen ist der Mechanismus dieser restriktiven EPO-Expression bislang aber unklar und ist daher Gegenstand intensiver Forschung, insbesondere im Hinblick auf die hohen Kosten, die klinisch bei der Behandlung mit rekombinantem Erythropoietin entstehen.In dieser Arbeit zeigten 9 (ca. 40%) der 22 untersuchten, renalen Tumorgewebsproben im RNase Protection Assay (RPA) eine EPO-Expression. Aus diesen 22 Tumorgewebsproben wurden primaere Zell-Linien etabliert, die, ergaenzt mit weiteren, extern gewonnenen Zellkulturen, 24 Zell-Linien fuer diese Arbeit zur Verfuegung stellten. Im Immunoblot konnte hier das Muster einer konstitutiven Expression der HIFa-Isoformen bei einer deutlichen Ueberzahl (17/24) nachgewiesen werden. Damit wurde indirekt eine Funktionseinschraenkung im pVHL angenommen, zudem schienen die Vorraussetzungen fuer eine EPO-Expression erfuellt. Trotzdem konnte im RPA bei keiner dieser Zell-Linien eine EPO-Expression gefunden werden. Dies war insofern unerwartet, als dass die 40% EPO-positiven Tumore auch eine EPO- Expression in der Zell-Linie hatten vermuten lassen, die notwendige HIF- Aktivierung gezeigt war und vorherige Arbeitsgruppen das erfolgreiche Etablieren einer stabil EPO-exprimierenden Zell-Linie beschrieben hatten. Kontraer zu diesem Ergebnis zeigte der Nachweis im RPA der CA-9 (Carboanhydrase-9) mRNA-Expression in denselben Zell-Linien eine HIF-1 konforme Expression.Bei der Abklaerung moeglicher Ursachen konnte mittels in- situ-Hybridisierung bestaetigt werden, dass die Tumorzellen Ort der EPO- Expression waren, aus denen die Zell-Linien etabliert wurden. Einen weiteren Versuch, eine EPO-exprimierende Zell-Linie zu etablieren, schien die in der Literatur beschriebene familiaere Polyzythaemie mit der Chuvash -Mutation des VHL-Gens zu erlauben. Aber auch nach stabiler Transfektion der bereits publizierten Zell-Linie RCC-4 mit dieser Chuvash -Mutation konnte unter in vitro Bedingungen keine EPO-Expression detektiert werden. Es ist daher festzuhalten, dass das Vermoegen, EPO zu exprimieren, offensichtlich in vitro verloren geht trotz Aktivierung des notwendigen HIF- Transkriptionskomplexes. Auch das Erzeugen eines stabilen VHL-Transgens konnte in vitro keine EPO-Expression hervorrufen. Auf die EPO-Expression in vitro scheinen daher weitere, bislang unbekannte Faktoren repressiven Einfluss auszuueben. Ob diese auch an der ausserordentlich gewebs- und zellspezifischen Expression in vivo beteiligt sind, bedarf weiterer Forschung.Erythropoietin (Epo) is the single most important stimulatory factor for erythropoiesis thereby directly involved in oxygen supply of the peripheral tissue. Physiologically, its gene expression is restricted to a small number of cells in the kidney. Dysregulation of Epo expression can be observed in certain diseases. Knowledge of the underlying molecular mechanisms will contribute to better understanding, perhaps even enable utilization of the system. So far it is well-established that induction of EPO requires the activation of HIF (hypoxia-inducible factor). HIF is targeted by the protein von-Hippel-Lindau (pVHL) which leads to its proteasomal degradation under normoxic conditions. Despite the advanced knowledge of oxygen sensing and regulation of HIF, the precise mechanism of the restricted Epo expression and the frequency of its expression in renal cell carcinoma remains unclear. 22 renal carcinomas were therefore investigated for their Epo expression using RNase protection assay from which about 40% showed an Epo expression. In addition 24 primary cell lines were analyzed which had been established from the renal carcinomas or were generated externally. Immunoblotting revealed a constitutive expression of HIFa in most cell lines (17/24) putatively associated with pVHL inactivation. Epo expression could not be observed in any primary cell line. This was unexpected since about 40% of the original tumor tissues had shown an Epo expression. In contrast, detection of CA-9 (carbonic anhydrase-9) expression, another HIF target gene, in the primary cell lines confirmed that HIF was activated. In order to address this absence of Epo expression in the cell lines in-situ- hybridization was performed. It was confirmed that the tumor cells were responsible for Epo production from which the primary cell lines had been generated. Stable transfection of RCC-4 cells with the Chuvash -mutation of the VHL gene which is related to congenital polycythemia could not establish an Epo producing cell line. It can be concluded from these data that HIF activation is necessary for Epo expression, but this is clearly not sufficient. Furthermore, expression of Epo in tumour tissues does not translate into cell cultures. Stable transfection of the Chuvash -mutation could not induce Epo expression in RCC-4 cells, therefore does not represent a gain-of-function situation. This implies that factors unknown up to now repress Epo expression in vitro and in vivo, which are most likely different. If those are involved in the tissue and cell specific Epo expression in vivo needs further to be investigated

Similar lymphocytic infiltration pattern in primary breast cancer and their corresponding distant metastases

Tumor infiltrating lymphocytes in primary breast cancer (TIL) are acknowledged measures of disease free survival (DFS) in adjuvant and neoadjuvant settings. Little is known about the biology of metastasis infiltrating lymphocytes (mTIL) although the local immunity of the metastatic site may critically influence the infiltrate composite. To address this question we compared mTIL with their matched TIL in 87 breast cancer patients and their corresponding distant metastasis at four different anatomical locations. Sections of surgical specimen were immunohistochemically analyzed for CD4, CD8 and CD20 positive lymphocytes in three different tumor compartments: intratumoral lymphocytes (iTIL) defined as lymphocytes in direct contact with breast cancer cells, stromal lymphocytes (sTIL) located within the intratumoral stromal tissue and invasive-margin lymphocytes (imTIL). Overall we found fewer (p < 0.001) mTIL than TIL. Within the tumor compartments imTIL were more frequent than sTIL and iTIL both within metastases and the matched primary tumors (p < 0.001). CD4+ T cells were more numerous than CD8+ T cells and CD20+ B cells (p < 0.001). There was a similar pattern in primary tumors and their corresponding metastasis. Only patients with brain metastases differed from the others displaying less CD20+ B cells at the infiltrative margin of the primary tumor (p < 0.05). In summary, mTIL were significantly reduced within metastases but still mirrored the infiltrate pattern of the primary tumor, interestingly regardless of the metastatic anatomical locations investigated. Our results suggest that the primary tumor assigns the infiltrating lymphocyte pattern resumed at the metastatic site