Evaluation of geno- and phenotypic alterations in Luminal B breast cancer using tumor mice (TM) and humanized tumor mice (HTM)

The breast cancer subtype Luminal B is diagnosed in 20% of all breast cancer cases whereas only 50 % of the patients are still alive 5 years after the first diagnosis. Despite the advances in treatment, patients suffering from Luminal B breast cancer frequently experience a relapse or develop distant metastases. Besides the current strategy of hormone-receptor-positivity, proliferation indices, grading, and gene signature assays to categorize the Luminal breast cancer patients into high and low-risk groups, there is still a lack of appropriate markers that reliably predict events of recurrence. Overall, this thesis aims to identify biomarkers that are associated with aggressivity, cell dissemination and/or metastases formation. Importantly these markers might contribute to the therapy decision if Luminal B breast cancer patients need a chemotherapeutic intervention or not. Therefore, the primary Luminal B patient samples were analyzed and PDX models were generated by the transplantation of primary Luminal B patient samples into NSG mice, the so-called tumor mouse (TM). Additionally, humanized Luminal B tumor mice (HTM) were generated and assessed under the influence of the human immune system. The phenotypic analysis of the primary patient samples revealed that a high expression of CD24 in Luminal B breast cancer patients differs from Luminal A breast cancer patients. The occurrence of MICs (CD44+/cMET+/CD47+) in the high-risk Luminal B tumors (patients that died, suffered from a relapse, or when the PDX model was successful) compared with low-risk Luminal B tumors (patients that are alive, without a relapse, and where the PDX model failed) could serve as a marker for the identification of high-risk Luminal B breast cancer patients. Remarkably, tumor cells of lung metastases differed phenotypically to those of the primary tumor, showing an increased CD44 and cMET expression in the TM, as well as in the patient metastases (e.g. pleural effusion and ascites). Enhanced expression of cMET and CD44 in Luminal B metastases were determined to be independent of the absence or the presence of a human immune system. Moreover, an increased CD4/CD8 ratio was determined as an indicator of a high-risk Luminal B tumor. However, the most important finding was the dependence of MDM2 amplification to form highly aggressive tumors accompanied by the high probability for metastatic spread in Luminal B TM and HTM. When MDM2 was amplified in tumors, the metastases preferentially were found in the lung of the PDX model, and DTCs in the bone marrow. This means that an amplification of MDM2 in Luminal breast cancer characterizes the patients as high-risk patients. These findings were confirmed in vitro by a MDM2 knockdown experiment, showing a p53 mediated mechanism of apoptosis and cell proliferation. Targeting MDM2 by AMG232 inhibition revealed increased apoptosis and reduced proliferation, which demonstrated the potential clinical relevance. TP53 mutation was also detected as a high-risk marker in Luminal B TM as this alteration in the primary tumor promoted BM DTCs. MDM4 amplification was verified to promote metastatic spread into various organs, such as the lung, the liver, the brain, and the BM, and subclassifies the tumor as a high-risk tumor. All the determined genomic alterations of MDM2, p53, and MDM4 regulate each other, which shows the importance of the pathway for high-risk Luminal B breast cancer. Single cell sequencing revealed one cluster formation of primary tumor with specific genomic losses and gains and another cluster mainly formed by DTCs. The differences in copy number profiles were preferentially shown by DTCs that derived from HTM PDX but not from TM, implicating a selection pressure in the periphery potentially evoked by human immune cells. Moreover, a selection determined by the bone marrow niche, which is altered by human immune cells in the HTM, could enable DTCs with a special genetic profile to colonize. The low immunogenicity of Luminal B tumors was demonstrated in primary patient samples and in the HTM, rendering the Luminal B HTM PDX as an adequate model to analyze Luminal B breast cancer. These models could be useful for preclinical immune-modulatory studies in Luminal B breast cancer in the future. In summary, we showed the suitability of Luminal B PDX and humanized PDX models that are able to identify geno- and phenotypic markers that predict a high potential for metastatic spread and aggressiveness of the tumor. However, prospectively further studies on MDM2 amplification and MDM2 expression in Luminal B breast cancer have to be validated in large patient cohorts. Further clinical studies should determine if breast cancer patients with genetic MDM2/MDM4/TP53 predisposition might additionally benefit from cytotoxic intervention or from specific MDM2 targeting (e.g., by MDM2 inhibitors)

mdm2 gene amplification is associated with luminal breast cancer progression in humanized PDX mice and a worse outcome of estrogen receptor positive disease

Estrogen receptor-positive breast cancer is a highly prevalent but heterogeneous disease among women. Advanced molecular stratification is required to enable individually most efficient treatments based on relevant prognostic and predictive biomarkers. First objective of our study was the hypothesis-driven discovery of biomarkers involved in tumor progression upon xenotransplantation of Luminal breast cancer into humanized mice. The second objective was the marker validation and correlation with the clinical outcome of Luminal breast cancer disease within the GeparTrio trial. An elevated mdm2 gene copy number was associated with enhanced tumor growth and lung metastasis in humanized tumor mice. The viability, proliferation and migration capacity of inherently mdm2 positive breast cancer cells in vitro were significantly reduced upon mdm2 knockdown or anti-mdm2 targeting. An mdm2 gain significantly correlated with a worse DFS and OS of Luminal breast cancer patients, albeit it was also associated with an enhanced preoperative pathological response rate. We provide evidence for an enhanced Luminal breast cancer stratification based on mdm2. Moreover, mdm2 can potentially be utilized as a therapeutic target in the Luminal subtype

Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell Lines In Vitro and in Immunodeficient and Humanized Tumor Mice

Programmed death ligand 1 (PD-L1) expression is an efficient strategy of tumor cells to escape immunological eradiation. However, only little is known about the factors that affect the cellular expression levels. Here we assessed the PD-L1 expression on different breast cancer cell lines under standard in vitro culture conditions and as a function of Epirubicin or Paclitaxel treatment. Moreover, we evaluated the expression in immunodeficient tumor mice as well as in humanized tumor mice (i.e., in the presence of a human immune system). We found highest PD-L1 levels in JIMT-1 and MDA-MB-231 cells. Epirubicin treatment caused a decrease and Paclitaxel treatment an increased PD-L1 expression in MDA-MB-231 cells. In addition, we identified nuclear PD-L1 in MDA-MB-231 cells. All in vivo transplanted breast cancer cell lines downregulated PD-L1 expression compared to their in vitro counterpart. Neither the gene copy number nor the presence of human immune system in humanized tumor mice had an effect on the PD-L1 content. We demonstrate that the degree of PD-L1 expression amongst breast cancer cell lines varies considerably. In addition, cytotoxic treatments and other extrinsic parameters differentially affect the expression. Hence, further investigations including in vivo evaluations are necessary to understand PD-L1 regulation for advanced breast cancer stratification

CCR7 Is Important for Mesangial Cell Physiology and Repair

The homeostatic chemokine receptor CCR7 serves as key molecule in lymphocyte homing into secondary lymphoid tissues. Previous experiments from our group identified CCR7 also to be expressed by human mesangial cells. Exposing cultured human mesangial cells to the receptor ligand CCL21 revealed a positive effect on these cells regarding proliferation, migration, and survival. In the present study, we localized CCR7 and CCL21 during murine nephrogenesis. Analyzing wild-type and CCR7 deficient (CCR7(-/-)) mice, we observed a retarded glomerulogenesis during renal development and a significantly decreased mesangial cellularity in adult CCR7(-/-) mice, as a consequence of less mesangial cell proliferation between embryonic day E17.5 and week 5 postpartum. Cell proliferation assays and cell-wounding experiments confirmed reduced proliferative and migratory properties of mesangial cells cultured from CCR7(-/-) kidneys. To further emphasize the role of CCR7 as important factor for mesangial biology, we examined the chemokine receptor expression in rats after induction of a mesangioproliferative glomerulonephritis. Here, we demonstrated for the first time that extra- and intraglomerular mesangial cells that were CCR7-negative in control rats exhibited a strong CCR7 expression during the phase of mesangial repopulation and proliferation