Brain metastases (BM) have a 90% mortality rate within 4-12 months of diagnosis because they are mainly treated palliatively. The Singh Lab uses patient-derived BM samples in clinically relevant xenograft models of BM that recapitulate BM progression as seen in human patients. The objectives of my thesis were twofold: to understand whether targeting pre-metastatic cells could intercept BM formation, and whether macro-metastatic BM tumors could respond to immunotherapy as an alternative to palliative care. To intercept BM formation, I mined a Connectivity Map analysis on pre-metastatic BM gene expression signatures and identified a key regulator of the pre-metastatic process. Pharmacological and genetic perturbation of this target attenuates BMIC proliferation in vitro and slows down the formation of BM in vivo, highlighting a targetable metabolic vulnerability in BM. This work has inspired a preclinical drug development program focused on creating inhibitors with enhanced brain permeation for clinical translation. To identify whether macro-metastatic tumors respond to immunotherapy, we used a chimeric antigen receptor (CAR) T-cell to target the glycoprotein CD133, whose expression correlates with disease progression, recurrence, chemo- and radio-resistance and overall poor prognosis. We saw significant impediment in tumor progression and significant survival advantage in tumor-bearing mice after a single dose. Together, I propose that prophylaxis should be the focus of BM research moving forward and that immunotherapies should be considered where BM diagnosis precedes the chance for interception. The development of a drug that can eradicate pre-metastatic cells has the potential to prevent BM in at-risk cancer patients, while the development of a CD133-targetting immunotherapy can dramatically improve the prognosis of patients who would otherwise be limited to palliative care. / Thesis / Doctor of Philosophy (PhD) / Brain metastases have a 90% mortality rate within one year of diagnosis because they are largely untreatable. The objectives of this research were twofold: to understand whether we can prevent BM, and to understand whether developed metastatic brain tumors respond to immunotherapy. We discovered that inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH) prevents the growth of brain metastases cells. This inspired an ongoing drug development program aiming to develop IMPDH inhibitors as drugs that prevent metastatic brain cancer. To identify whether metastatic brain tumors respond to immunotherapy, we used a CAR-T cell immunotherapy to target the glycoprotein CD133 and found that tumor growth was delayed or completely blocked after a single dose. Based on these results, we propose that therapeutics that prevent the formation of brain metastases are a promising strategy to combat this deadly disease. Furthermore, we have shown that immunotherapies are a viable strategy for the treatment of brain metastases.

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Hypofractionated conformal stereotactic radiotherapy (HCSRT) has been used for the treatment of AVMs at the Umeå University Hospital since 1986. From this year and onwards an increasing number of patients with single or oligo brain metastases have also been treated using this technique. In paper I we have retrospectively evaluated our treatment results of AVMs in terms of obliteration and complications. The rates of obliteration and complications seem to be comparable with SRS even if the AVM volumes in our series were larger than in most series with SRS. In paper II we have retrospectively evaluated the results in terms of local control, survival and complications in two groups of patients with single or oligo brain metastases. One group was treated with HCSRT alone and the other group was treated with whole brain radiotherapy in combination with a stereotactic boost. Controversy still exists concerning the benefit of additional use of WBRT in combination with stereotactic irradiation. The survival times were equal in the two groups and no significant difference in local control was observed. The omission of WBRT seems to carry a higher risk for development new brain metastases distant from the irradiated area. In paper III we report the treatment results in a subgroup of AVMs treated with a combination of embolisation and HCSRT. We also focus on the reduction of vascular density within the nidus of an AVM and propose a method to digitally compare images and more objectively assess a reduction in vascular density following embolisation. Obliteration rates seem comparable with other series using a combination of SRS and embolisation even if our rate of complications was higher than what is usually reported. Using luminescence as measure of vascular density all AVMs seemed to be less dense after embolisation. Treatment accuracy in terms of reproducibility of the isocenter in consecutive treatment sessions is crucial in fractionated radiotherapy. In paper IV we have radiologically evaluated the reproducibility of the isocenter in successive treatment sessions using the non invasive relocatable Fixster frame. There was a high degree of reproducibility and only small errors that most likely is of no clinical importance. A reliable dose plan is equally important as a tool to predict the dose delivered inside and outside the target volume. In paper V we have evaluated the reliability of treatment plans in HCSRT for targets of different geometry and size. A liquid ion chamber and gel dosimeter was used for assessment of dose distribution and absorbed dose. The doseplanning system proved to be accurate in predicting the absorbed dose and dose distribution for the different targets.

Conclusion: Surgery should be considered whenever possible. This means that the patient has to be in good clinical condition (Karnofsky performance score > 70), the extracerebral metastases should be stable, the number of cerebral lesions should not exceed more than 3 seedings, and the age of the patient should be below 70 years. Since brain metastases are usually well circumscribed, complete extirpation seems to be possible. Postoperative MRI should be demanded in order to confirm complete extirpation. Additional radiotherapy is indicated in case of subtotal resection of a single lesion and in multiple lesions. In single brain metastasis a prospective randomized trial is necessary to prove whether conventional radiotherapy is essential after surgery in the primary treatment of the tumors or can be delayed until cerebral lesions recur. Radiosurgery is an alternative to surgery in the treatment of metastasis. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Brain metastases are treated with surgery, radiotherapy, radiosurgery, and chemotherapy. In this review, recently published studies concerning different treatment strategies are presented with respect to solitary lesions, multiple metastases, and recurrent tumor growth. Selection criteria for the appropriate therapy are: control of the primary tumor, extent of extracerebral metastases, time interval between diagnosis of the primary tumor and the development of cerebral lesions, number of cerebral metastases, Karnofsky performance scale score, and age. Treatment approaches were evaluated with respect to median survival time and quality of life. A singular brain metastasis can be treated with surgery or with radiosurgery. Especially when the primary tumor is under control, there are few extracerebral lesions which are stable, the Karnofsky performance scale score is above 70, the lesion is larger than 3 cm in diameter and surgically accessible surgery is the treatment of choice. Postoperative adjuvant radiotherapy may delay relapse. Median survival time ranges between 10 to 18 months. Radiosurgery can be applied in lesions smaller than 3 cm in diameter and is the treatment of choice in lesions which are surgically not accessible. Multiple metastases are treated either by conventional radiotherapy, radiosurgery or surgery. Commonly, no more than 3 lesions are approached by either surgery or radiosurgery. Median survival time ranges between 6 to 9 months for both treatment concepts, but without therapy only is 4–6 weeks. According to the clinical and neurological condition of the patient, recurrent brain metastases can be treated by operation, reirradiation, or radiosurgery. The efficacy of chemotherapy depends on the chemosensitivity of the primary tumor and the ability to penetrate the blood-brain barrier. Long-term survivors with cancer disease encourage to perform active treatment strategies. / Hirnmetastasen werden durch Operation, Ganzhirnbestrahlung, Radiochirurgie und Chemotherapie behandelt. In dieser Übersichtsarbeit werden kürzlich publizierte Studien bezüglich der Therapiekonzepte für solitäre Läsionen, multiple Metastasen und Tumorrezidive vorgestellt. Auswahlkriterien für eine angemessene Behandlung sind: Kontrolle des Primärtumors, Ausmaß der extrakraniellen Metastasen, Zeitintervall zwischen Diagnose des Primärtumors und dem Auftreten der Hirntumoren, Anzahl der zerebralen Metastasen, Karnofsky-Performance-Scale-Score und Lebensalter. Behandlungskonzepte wurden nach der medianen Überlebenszeit und Lebensqualität ausgewertet. Singuläre Hirnmetastasen können operativ oder radiochirurgisch behandelt werden. Insbesondere wenn der Primärtumor unter Kontrolle ist, wenige extrazerebrale Läsionen bestehen und diese stabil sind, der Karnofsky-Performance-Scale-Score über 70 ist, die Tumoren größer als 3 cm im Durchmesser und chirurgisch erreichbar sind, ist die Operation die Methode der Wahl. Postoperative adjuvante Strahlentherapie kann erneute Progression verzögern. Die mediane Überlebenszeit liegt zwischen 10 und 18 Monaten. Für Läsionen, die kleiner als 3 cm sind und chirurgisch nicht erreicht werden können, ist die Radiochirurgie die Therapie der Wahl. Multiple Metastasen können durch konventionelle Ganzhirnbestrahlung, Radiochirurgie oder Operation behandelt werden. Im allgemeinen werden nicht mehr als 3 Herde operativ oder radiochirurgisch angegangen. Die mediane Überlebenszeit liegt bei beiden Therapieformen zwischen 6 und 9 Monaten, ohne Behandlung hingegen bei nur 4–6 Wochen. Entsprechend dem klinischen und neurologischen Zustand der Patienten können Rezidive von Hirnmetastasen durch chirurgische Entfernung, erneute Bestrahlung oder durch Radiochirurgie therapiert werden. Die Wirkung der Chemotherapie hängt von der Chemosensitivität des Primärtumors und der Durchlässigkeit der Blut-Hirn-Schranke für das Chemotherapeutikum ab. Langzeitüberleber motivieren zu aktiven Behandlungsstrategien. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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Yes / Recent evidence suggests that the ion channel TRPA1 is implicated in lung adenocarcinoma (LUAD) where its role and mechanism of action remain unknown. We have previously established that the membrane receptor FGFR2 drives LUAD progression through aberrant protein-protein interactions mediated via its C-terminal proline rich motif. Here, we report that the N-terminal ankyrin repeats of TRPA1 directly bind to the C-terminal proline rich motif of FGFR2 inducing the constitutive activation of the receptor, thereby prompting LUAD progression and metastasis. Furthermore, we show that upon metastasis to the brain, TRPA1 gets depleted, an effect triggered by the transfer of TRPA1-targeting exosomal microRNA (miRNA-142-3p) from brain astrocytes to cancer cells. This downregulation, in turn, inhibits TRPA1-mediated activation of FGFR2 hindering the metastatic process. Our study reveals a direct binding event and characterizes the role of TRPA1 ankyrin repeats in regulating FGFR2-driven oncogenic process; a mechanism that is hindered by miRNA-142-3p. / Faculty of Biological Sciences at the University of Leeds, Wellcome Trust Seed Award, Royal Society Research Grant RG150100, MR/K021303/1, Swedish Research Council (2014-3801) and the Medical Faculty at Lund University.

Brain metastases (BMs) present a formidable obstacle across various primary cancer types, notably small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), melanomas, and breast cancers. In this investigation, we aim to evaluate the potential of genotype-guided targeted therapy while addressing the challenges of co-existing genomic alterations frequently encountered in BMs. This research explores the efficacy of adagrasib (MRTX849), a KRAS G12C inhibitor, and abemaciclib, a CDK 4/6 inhibitor, both individually and in combination against BMs originating from NSCLC cell lines harboring KRAS G12C and CDKN2A mutations. Utilizing a diverse array of methodologies encompassing cell viability assays, cell death assays, western blot analyses, and in vivo xenograft models, we elucidate both the therapeutic potential and underlying mechanisms. Distinct responses to adagrasib and abemaciclib monotherapies were observed across two different cell lines, underscoring the necessity for tailored treatment strategies. While adagrasib exhibited variable efficacy, abemaciclib consistently inhibited CDK 4/6 activity. Notably, the combination therapy demonstrated synergistic effects, suggesting a promising approach for enhanced therapeutic outcomes. Our findings from both in vitro assays and western blot analyses corroborate targeted pathway inhibition, although the observed pathway reactivation underscores the importance of optimizing dosing strategies. In vivo studies further support our in vitro findings, demonstrating efficacy but also raising concerns regarding toxicity with combination therapy. Pharmacokinetic / pharmacodynamic (PK/PD) analyses underscore potential advantages of combination therapy in terms of systemic exposure and brain penetration. Despite histological evidence of therapeutic effects, discrepancies between in vivo and in vitro caspase-dependent apoptosis results highlight the complexity of tumor biology and the challenges of translation. By Focusing on personalized treatment approaches and addressing therapeutic hurdles, this work establishes the foundation for clinical investigation in advancing the management of BMs and improving treatment outcomes in NSCLC patients.

Die Dissertation hat sich mit der Aufgabe befasst, durch Kombination von IR-Spektroskopie und chemometrischen Auswertungsalgorithmen eine Differenzierung und Klassifizierung von Hirnmetastasen-Dünnschnitten zu erreichen. Die Untersuchungen konzentrieren sich dabei auf jene fünf Primärtumoren, die besonders oft Metastasen im Gehirn bilden. Das sind kolorektale Karzinome, Mammakarzinome, maligne Melanome, Nierenzellkarzinome und Bronchialkarzinome. Metastasen tragen die molekularen Informationen der Gewebezellen des Primärtumors in sich. Die Anwendung von IR-spektroskopischen Methoden stellt deshalb einen innovativen Ansatz zur Identifikation des Primärtumors von Hirnmetastasen dar, da die Spektren einem molekularen Fingerabdruck entsprechen. Als Klassifizierungsalgorithmen wurden SIMCA (soft independent modeling of class analogies) und ANN (artificial neural networks) herangezogen. Die Entwicklung der Klassifizierungsverfahren gliederte sich in drei Teile. Im ersten Teil wurden Trainingsmodelle mit den ausgewählten homogenen Bereichen der Metastasengewebeschnitte erstellt und an unabhängigen Daten weiterer Proben bekannter und unbekannter Organherkunft getestet. Im zweiten Teil wurden die Modelle mit Hilfe homogener Tumorzelllinien optimiert und auf die Zuordnung der Hirnmetastasen zu den Primärtumoren angewandt. Eine zweistufige Klassifizierungsstrategie gewährleistet damit eine Genauigkeit der Klassifizierung von über 80%.

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