TU Darmstadt / ULB / TUbiblio

The influence of radio-immunotherapy on the tumor microenvironment of breast-to-brain metastasis and the investigation of novel adjuvant therapies

Niesel, Katja Anne (2021):
The influence of radio-immunotherapy on the tumor microenvironment of breast-to-brain metastasis and the investigation of novel adjuvant therapies. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.26083/tuprints-00018577,
[Ph.D. Thesis]

Abstract

With ongoing progress in cancer research and continuously improving treatment strategies for primary tumors, the incidence of brain metastasis is steadily increasing. The treatment options for brain metastasis patients, however, are limited and only prolong survival for a short duration. With the advent of immunotherapies, the cancer field was revolutionized. Checkpoint inhibitors, which reactivate T cell responses against cancer cells, show promising results even for aggressive cancers such as advanced metastatic melanoma. The brain was regarded as an immune privileged site for a long time. However, only recently a classical lymphatic system has been revealed in the brain. Moreover, the central nervous system harbors a greater variety of immune cells than previously assumed. Therefore, immunotherapies including checkpoint inhibitors, also gain interest for the treatment of brain metastases. To date, most research in the cancer field focusses on highly immunogenic cancer entities, whereas tumors of low immunogenicity such as breast cancer are less well investigated, as they are thought to be resistant to checkpoint inhibition. The development of strategies to convert 'cold' tumors of low immunogenicity into 'hot' tumors with a pro-inflammatory tumor microenvironment is of great interest, as they potentially sensitize highly immune suppressive tumor microenvironments to immune checkpoint inhibition. One strategy, that has shown promising results for different cancer entities is the combination of checkpoint inhibition with radiotherapy. The efficacy of this combination is investigated in clinical trials, including the treatment of brain metastasis. Current research focusses mostly on melanoma and lung cancer derived brain metastasis, and only little information is available on the efficacy of radio-immunotherapy in the treatment of breast cancer brain metastasis. Therefore, the aim of this thesis was to investigate if standard of care radiotherapy can sensitize breast cancer brain metastasis to immune checkpoint inhibition. In this context, the tumor microenvironment of the murine breast cancer brain metastasis model 99LN-BrM was investigated in detail. The purpose was to obtain an overview of proportions of cells, counteracting T cell responses, versus cells that are crucial for efficient checkpoint inhibition. It was confirmed that the tumor microenvironment of 99LN BrM is a typical 'cold' microenvironment dominated by myeloid cells. However, cell types, crucial for checkpoint blockade, such as T cells and dendritic cells were identified, too. The next step was the examination of the influence of ionizing radiation on brain homing breast cancer cells and on the immune cell composition in 99LN-BrM. It was revealed that brain homing breast cancer cells increasingly express inflammatory markers, such as TNFα and IL1β, after in vitro irradiation. In a preclinical trial, the treatment of 99LN BrM mice with fractionated whole brain radiotherapy, did not lead to increased recruitment of potentially immune suppressive cell types, such as blood borne myeloid cells or regulatory T cells. Moreover, radiosensitive cell types, crucial for efficient checkpoint inhibition, such as T cells and dendritic cells were not depleted. On the contrary, the infiltration of cytotoxic CD8+ T cells into 99LN-BrM lesion was increased by fractionated whole brain radiotherapy. To obtain a deeper understanding of the T cell compartment in 99LN-BrM, TCRβ-profiling was performed next. These data reveled, that T cells in 99LN-BrM lesions and in CNS draining lymph nodes, clonally expand, indicating prior tumor directed T cell activation. However, a negative correlation of T cell expansion with brain metastasis volume was observed. This indicates progressive inhibition of T cell responses, which was confirmed by in vivo T cell depletion experiments. The depletion of T cells in mice, injected with brain homing 99LN-BrM cells, did not shorten the time of brain metastasis onset. This demonstrates that T cell responses in the microenvironment of 99LN-BrM are sufficiently suppressed. A checkpoint axis, which often plays a crucial role in immune system evasion of cancer cells, is the PD 1/PD L1 axis. By expressing PD-L1, tumor cells can inhibit PD-1 positive T cells in the tumor microenvironment. Assessment of PD-L1 expression by brain homing breast cancer cell lines showed expression on the RNA- and protein level in vitro. In vivo analysis revealed that a high proportion of T cells in 99LN-BrM express PD-1, whereas PD-L1 is expressed by tumor cells, myeloid and T cells. Furthermore, analysis of the myeloid compartment demonstrated that a high proportion of infiltrating myeloid cells is PD L1 positive, which is not the case for brain resident microglia. In a preclinical trial, treatment of 99LN-BrM mice with anti-PD-1, whole brain radiotherapy or a combination of both exhibited superior efficacy of the radio-immunotherapy over the monotherapies. Tumor progression slowed down, which translated into significantly prolonged median survival. However, long term survival was not achieved. The flow cytometric and histological analysis of brain metastases from mice in the preclinical trial, revealed that only in the combination cohort, both the infiltration of CD4+ and CD8+ T cells was enhanced in the brain metastasis lesions. At the same time, increased infiltration of blood-borne PD-L1+ myeloid cells, especially monocyte derived macrophages, was observed. Moreover, this infiltration was most prominent in the combination treatment group and indicates a crucial role of monocyte derived macrophages in acquired resistance to radio-immunotherapy. Furthermore, the in vitro assessment of T cell inhibitory capacity of 99LN-BrM conditioned microglia versus monocyte derived macrophages, revealed that the latter inhibit T cells more efficiently. To develop strategies to induce long term efficacy, macrophages were targeted pharmacologically, in addition to radio-immunotherapy. One strategy included the inhibition of the chemokine receptor CXCR4, expressed by macrophages, with the CXCR4 inhibitor AMD3100. This approach aimed at inhibiting the recruitment of monocyte derived macrophages to brain metastases. The second approach was aimed at targeting all macrophages by inhibiting CSF1R, the receptor to the macrophage survival factor CSF1. In vitro, the components of both signaling pathways were expressed by brain homing breast cancer cell lines. However, in vivo, both macrophage targeting strategies did not induce long term efficacy of radio-immunotherapy with anti-PD-1. Analysis of 99LN-BrM lesions revealed that CXCR4 inhibition failed to inhibit the recruitment of monocyte derived macrophages and even increased the infiltration of 99LN BrM with PD L1+ immune cells. The pharmacological inhibition of CSF1R not only led to the depletion of most macrophages in brain metastasis, including microglia, but also significantly decreased T cell infiltration, which is crucial for the efficacy of checkpoint inhibition. To induce long term efficacy in the future, a deeper understanding of myeloid immune suppressive cells in breast cancer brain metastasis is crucial. Additionally, treatment strategies, targeting the recruitment of blood borne myeloid cells specifically, while sparing other immune cells, need to be developed.

Item Type: Ph.D. Thesis
Erschienen: 2021
Creators: Niesel, Katja Anne
Status: Publisher's Version
Title: The influence of radio-immunotherapy on the tumor microenvironment of breast-to-brain metastasis and the investigation of novel adjuvant therapies
Language: English
Abstract:

With ongoing progress in cancer research and continuously improving treatment strategies for primary tumors, the incidence of brain metastasis is steadily increasing. The treatment options for brain metastasis patients, however, are limited and only prolong survival for a short duration. With the advent of immunotherapies, the cancer field was revolutionized. Checkpoint inhibitors, which reactivate T cell responses against cancer cells, show promising results even for aggressive cancers such as advanced metastatic melanoma. The brain was regarded as an immune privileged site for a long time. However, only recently a classical lymphatic system has been revealed in the brain. Moreover, the central nervous system harbors a greater variety of immune cells than previously assumed. Therefore, immunotherapies including checkpoint inhibitors, also gain interest for the treatment of brain metastases. To date, most research in the cancer field focusses on highly immunogenic cancer entities, whereas tumors of low immunogenicity such as breast cancer are less well investigated, as they are thought to be resistant to checkpoint inhibition. The development of strategies to convert 'cold' tumors of low immunogenicity into 'hot' tumors with a pro-inflammatory tumor microenvironment is of great interest, as they potentially sensitize highly immune suppressive tumor microenvironments to immune checkpoint inhibition. One strategy, that has shown promising results for different cancer entities is the combination of checkpoint inhibition with radiotherapy. The efficacy of this combination is investigated in clinical trials, including the treatment of brain metastasis. Current research focusses mostly on melanoma and lung cancer derived brain metastasis, and only little information is available on the efficacy of radio-immunotherapy in the treatment of breast cancer brain metastasis. Therefore, the aim of this thesis was to investigate if standard of care radiotherapy can sensitize breast cancer brain metastasis to immune checkpoint inhibition. In this context, the tumor microenvironment of the murine breast cancer brain metastasis model 99LN-BrM was investigated in detail. The purpose was to obtain an overview of proportions of cells, counteracting T cell responses, versus cells that are crucial for efficient checkpoint inhibition. It was confirmed that the tumor microenvironment of 99LN BrM is a typical 'cold' microenvironment dominated by myeloid cells. However, cell types, crucial for checkpoint blockade, such as T cells and dendritic cells were identified, too. The next step was the examination of the influence of ionizing radiation on brain homing breast cancer cells and on the immune cell composition in 99LN-BrM. It was revealed that brain homing breast cancer cells increasingly express inflammatory markers, such as TNFα and IL1β, after in vitro irradiation. In a preclinical trial, the treatment of 99LN BrM mice with fractionated whole brain radiotherapy, did not lead to increased recruitment of potentially immune suppressive cell types, such as blood borne myeloid cells or regulatory T cells. Moreover, radiosensitive cell types, crucial for efficient checkpoint inhibition, such as T cells and dendritic cells were not depleted. On the contrary, the infiltration of cytotoxic CD8+ T cells into 99LN-BrM lesion was increased by fractionated whole brain radiotherapy. To obtain a deeper understanding of the T cell compartment in 99LN-BrM, TCRβ-profiling was performed next. These data reveled, that T cells in 99LN-BrM lesions and in CNS draining lymph nodes, clonally expand, indicating prior tumor directed T cell activation. However, a negative correlation of T cell expansion with brain metastasis volume was observed. This indicates progressive inhibition of T cell responses, which was confirmed by in vivo T cell depletion experiments. The depletion of T cells in mice, injected with brain homing 99LN-BrM cells, did not shorten the time of brain metastasis onset. This demonstrates that T cell responses in the microenvironment of 99LN-BrM are sufficiently suppressed. A checkpoint axis, which often plays a crucial role in immune system evasion of cancer cells, is the PD 1/PD L1 axis. By expressing PD-L1, tumor cells can inhibit PD-1 positive T cells in the tumor microenvironment. Assessment of PD-L1 expression by brain homing breast cancer cell lines showed expression on the RNA- and protein level in vitro. In vivo analysis revealed that a high proportion of T cells in 99LN-BrM express PD-1, whereas PD-L1 is expressed by tumor cells, myeloid and T cells. Furthermore, analysis of the myeloid compartment demonstrated that a high proportion of infiltrating myeloid cells is PD L1 positive, which is not the case for brain resident microglia. In a preclinical trial, treatment of 99LN-BrM mice with anti-PD-1, whole brain radiotherapy or a combination of both exhibited superior efficacy of the radio-immunotherapy over the monotherapies. Tumor progression slowed down, which translated into significantly prolonged median survival. However, long term survival was not achieved. The flow cytometric and histological analysis of brain metastases from mice in the preclinical trial, revealed that only in the combination cohort, both the infiltration of CD4+ and CD8+ T cells was enhanced in the brain metastasis lesions. At the same time, increased infiltration of blood-borne PD-L1+ myeloid cells, especially monocyte derived macrophages, was observed. Moreover, this infiltration was most prominent in the combination treatment group and indicates a crucial role of monocyte derived macrophages in acquired resistance to radio-immunotherapy. Furthermore, the in vitro assessment of T cell inhibitory capacity of 99LN-BrM conditioned microglia versus monocyte derived macrophages, revealed that the latter inhibit T cells more efficiently. To develop strategies to induce long term efficacy, macrophages were targeted pharmacologically, in addition to radio-immunotherapy. One strategy included the inhibition of the chemokine receptor CXCR4, expressed by macrophages, with the CXCR4 inhibitor AMD3100. This approach aimed at inhibiting the recruitment of monocyte derived macrophages to brain metastases. The second approach was aimed at targeting all macrophages by inhibiting CSF1R, the receptor to the macrophage survival factor CSF1. In vitro, the components of both signaling pathways were expressed by brain homing breast cancer cell lines. However, in vivo, both macrophage targeting strategies did not induce long term efficacy of radio-immunotherapy with anti-PD-1. Analysis of 99LN-BrM lesions revealed that CXCR4 inhibition failed to inhibit the recruitment of monocyte derived macrophages and even increased the infiltration of 99LN BrM with PD L1+ immune cells. The pharmacological inhibition of CSF1R not only led to the depletion of most macrophages in brain metastasis, including microglia, but also significantly decreased T cell infiltration, which is crucial for the efficacy of checkpoint inhibition. To induce long term efficacy in the future, a deeper understanding of myeloid immune suppressive cells in breast cancer brain metastasis is crucial. Additionally, treatment strategies, targeting the recruitment of blood borne myeloid cells specifically, while sparing other immune cells, need to be developed.

Place of Publication: Darmstadt
Collation: ix, 133 Seiten
Divisions: 10 Department of Biology
10 Department of Biology > Synthetic Genetic Circuits
Date Deposited: 26 May 2021 08:02
DOI: 10.26083/tuprints-00018577
Official URL: https://tuprints.ulb.tu-darmstadt.de/18577
URN: urn:nbn:de:tuda-tuprints-185778
Referees: Süß, Prof. Dr. Beatrix ; Galuske, Prof. Dr. Ralf
Refereed / Verteidigung / mdl. Prüfung: 4 May 2021
Alternative Abstract:
Alternative abstract Language

Durch fortschreitende Entwicklungen in der Krebsforschung und Verbesserungen der Behandlungsoptionen zur Bekämpfung primärer Tumore steigt auch die Inzidenz von Hirnmetastasen. Die Therapiemöglichkeiten für Patienten mit Hirnmetastasen sind jedoch begrenzt und verlängern das Überleben nur kurzzeitig. Mit dem Aufkommen neuartiger Immuntherapien wurde das Feld der Krebsforschung revolutioniert. Checkpoint Inhibitoren, die T-Zellen gegen Krebszellen reaktivieren können, zeigen nach ersten Erkenntnissen Erfolge, sogar im Falle aggressiver Krebsarten wie fortgeschrittener Melanome und Lungenkarzinome. Das Gehirn wurde lange Zeit als immun privilegiert angesehen. Kürzlich wurde jedoch ein klassisches lymphatisches System im zentralen Nervensystem entdeckt. Außerdem ist die Vielfalt an Immunzellen größer als bisher angenommen. Aus diesem Grund, gewinnen Immuntherapien wie Checkpoint Inhibitoren an Bedeutung für die Behandlung von Patienten mit Hirnmetastasen. Der aktuelle Fokus der Forschung liegt auf hochgradig immunogenen Krebsentitäten. Tumore mit geringer Immunogenität wie Brustkrebs wurden bisher weniger intensiv erforscht, da hier von Resistenzen gegen Checkpoint Inhibitoren ausgegangen wird. Die Entwicklung von Strategien um sogenannte 'kalte' Tumore mit geringer Immunogenität in 'heiße' Tumore mit pro-inflammatorischem Tumormikromilieu umzuwandeln und sie somit für Checkpoint Inhibition zu sensibilisieren, ist von großem Interesse. Eine vielversprechende Strategie, die gute Ergebnisse in der Behandlung verschiedener Krebsarten zeigt, ist die Kombination von Checkpoint Inhibitoren mit Radiotherapie. Die Wirksamkeit dieser Kombination wird aktuell in klinischen Studien untersucht, auch in Bezug auf Hirnmetastasen. Dennoch liegt der Fokus auch im Falle der Kombinationstherapien hauptsächlich auf dem Melanom und Lungenkrebs, während Erkenntnisse zur Wirksamkeit von Radio-Immuntherapie gegen Brustkrebs-Hirnmetastasen sehr begrenzt sind. Aus diesem Grund war das Ziel dieser Arbeit zu untersuchen, ob Standard-Radiotherapie Brustkrebs-Hirnmetastasen für Checkpoint Inhibition sensibilisieren kann. In diesem Zusammenhang wurde das Tumormikromilieu des murinen Brustkrebs-Hirnmetastase Modells 99LN-BrM im Detail untersucht, mit dem Hintergrund, den Anteil T Zell hemmender Zelltypen im Gegensatz zu Zellen, die essenziell für Checkpoint Inhibition sind, zu bestimmen. Es konnte bestätigt werden, dass das Tumormikromilieu von 99LN-BrM zwar ein typisch 'kaltes' Milieu ist, das von myeloiden Zellen dominiert wird. Jedoch konnten auch Zelltypen, die für Checkpoint Inhibition essenziell sind, identifiziert werden. Im nächsten Schritt wurde der Einfluss von Bestrahlung auf Brustkrebszelllinien und auf die Komposition von Immunzellen in 99LN-BrM Hirnmetastasen untersucht. Es zeigte sich, dass Brustkrebszelllinien mit Hirntropismus nach in vitro Bestrahlung verstärkt inflammatorische Marker wie TNFα and IL1β exprimieren. Präklinischen Studien offenbarten, dass die Behandlung von 99LN-BrM Mäusen mit fraktionierter Ganzhirnbestrahlung nicht die Rekrutierung potenziell immun suppressiver Zelltypen wie peripherer myeloider Zellen oder regulatorischer T-Zellen verstärkt. Außerdem wurden radiosensitive Zelltypen wie T-Zellen und dendritische Zellen, die ausschlaggebend für die Wirkung von Checkpoint Inhibition sind, nicht depletiert. Im Gegenteil, fraktionierte Ganzhirnbestrahlung steigerte sogar die Infiltration von zytotoxischen CD8+ T-Zellen in 99LN BrM Läsionen. Um ein tieferes Verständnis des T-Zell Kompartiments in 99LN-BrM Läsionen zu erhalten, wurde TCRβ-Sequenzierung angewendet. Diese Analysen zeigten, dass T-Zellen in 99LN-BrM Läsionen und zervikalen Lymphknoten klonal expandieren, was ein Indikator für vorangehende T-Zellaktivierung ist. Jedoch wurde auch eine negative Korrelation von T-Zell Expansion mit Tumorvolumen beobachtet. Dies deutet auf eine fortschreitende Suppression der T Zellantwort hin, was mit in vivo T-Zell Depletions-Experimenten bestätigt werden konnte. Die Depletion von T-Zellen in Mäusen denen 99LN BrM Zellen intrakardial injiziert wurden, verringerte nicht die Zeit bis zur Entstehung der Hirnmetastasen. Dies weist auf eine effektive Suppression der T-Zellantwort in 99LN-BrM hin. Tumorzellen nutzen hierbei grundlegende Mechanismen der Regulation der T-Zellaktivität mittels Immuncheckpunkten aus. In diesem Zusammenhang spielt die PD-1/PD-L1-Achse eine zentrale Rolle. Durch die Expression von PD-L1 können Tumorzellen PD-1 positive T-Zellen im Tumormikromilieu hemmen. Die in vitro Analyse der PD-L1 Expression in Brustkrebszelllinien mit Hirntropismus zeigte, dass der Ligand auf RNA- und Proteinebene exprimiert wird. In vivo Analysen demonstrierten, dass ein hoher Anteil an T-Zellen in 99LN BrM Läsionen PD-1 exprimieren, während PD-L1 von Tumorzellen, myeloiden Zellen und T Zellen exprimiert wird. Die Analyse des myeloiden Kompartiments zeigte außerdem, dass ein hoher Anteil der infiltrierenden myeloiden Zellen PD-L1 positiv ist. Dies ist nicht der Fall bei den hirnresidenten Mikroglia. In einer präklinischen Studie von 99LN-BrM Mäusen mit anti-PD-1, Bestrahlung oder eine Kombination aus Beidem, zeigte die Radio-Immuntherapie erhöhte Wirksamkeit im Vergleich zu den Monotherapien. Die Tumorprogression wurde verzögert, was zu einem signifikant erhöhten medianen Überleben führte. Ein langfristiger Effekt konnte jedoch nicht erzielt werden. Durchflusszytometrie und histologische Analysen offenbarten, dass die Infiltration von CD4- als auch CD8-positiven T-Zellen, nur in der Kombinationsgruppe erhöht war. Zur selben Zeit verstärkte sich jedoch auch die Infiltration mit PD-L1 positiven peripheren myeloiden Zellen, besonders mit Knochenmarks-Makrophagen. Am ausgeprägtesten war diese Infiltration in der Kombinationsgruppe zu beobachten, was auf eine wichtige Rolle der peripheren Makrophagen in der Resistenzentwicklung gegen die angewendete Radio-Immuntherapie hindeutet. Außerdem ist die Fähigkeit von tumorkonditionierten Knochenmarks-Makrophagen, T-Zellen in vitro zu hemmen, größer im Vergleich zu tumorkonditionierten Mikroglia. Um Möglichkeiten zu finden Langzeiteffekte zu erzielen, wurden zusätzlich zur Radio-Immuntherapie mit anti-PD-1 Makrophagen pharmakologisch inhibiert. Eine Strategie beinhaltete die Inhibition des CXCR4 Rezeptors, der von Makrophagen exprimiert wird, durch den CXCR4 Inhibitor AMD3100. Dieser Ansatz sollte die Rekrutierung von Knochenmarks-Makrophagen zu den Hirnmetastasen hemmen. Der zweite Ansatz umfasste die Blockade von CSF1R, der Rezeptor des Zytokins CSF1, das wichtig ist für das Überleben von Makrophagen im Allgemeinen. In vitro Analysen bestätigten die Expression der Komponenten beider Signalwege in Brustkrebszelllinien. In präklinischen Studien konnten beide Strategien jedoch keine Langzeitwirkung der Radio-Immuntherapie induzieren. Die Analyse der 99LN-BrM Läsionen zeigte, dass die Inhibition von CXCR4 nicht die Rekrutierung von Knochenmarks-Makrophagen aufhalten konnte. Die Infiltration von PD L1 positiven Immunzellen wurde sogar verstärkt. Die pharmakologische Hemmung von CSF1R führte zwar zur Depletion der meisten Makrophagen in den Hirnmetastasen, Mikroglia eingeschlossen, sie führte jedoch auch zu einer signifikanten Verringerung der T Zellinfiltration, welche essenziell für die Wirksamkeit von Checkpoint Inhibition ist. Um in der Zukunft Langzeiteffekte zu erzielen, muss ein tiefergehendes Verständnis der Rolle myeloider immunsuppressiver Zellen in Brustkrebs-Hirnmetastasen erreicht werden. Außerdem müssen Strategien entwickelt werden, die spezifisch die Rekrutierung peripherer myeloider Zellen hemmen, während andere Immunzellen verschont bleiben.

German
Export:
Suche nach Titel in: TUfind oder in Google
Send an inquiry Send an inquiry

Options (only for editors)
Show editorial Details Show editorial Details