project 1) “CAFs – Paving The Way For New Cancer Therapies” funded by FWF Elise Richter Programme
approved amount (01.10.2016 – 30.09.2020):
€ 359.562
abstract:
In recent years the traditional view of cancer as a group of malignantly transformed cells has been greatly revised. The importance of the tumour vasculature, the immune system, the extracellular matrix as well as other cell types of the tumour stroma to promote tumour progression and metastasis have been demonstrated in many experimental studies. However, one cell type, which is in close contact to neoplastic cells throughout tumour progression, the fibroblast, has barely been characterized. Our knowledge of the role of resting and activated fibroblasts in cancer is still evolving, but there is now convincing evidence that the so-called cancer-associated fibroblasts (CAFs) differ from those associated with normal tissue. Only recently CAFs have been shown to play an important role in modulating malignant progression, and are therefore attractive targets for cancer therapy, especially since in contrast to cancer cells, CAFs rarely display genetic mutations. However, the origin of CAFs, their phenotypic and functional heterogeneity remain elusive. Importantly, we have recently demonstrated that skin dermis arises from two distinct fibroblast lineages, which have different functions in homeostasis and during regeneration, and thus, it is very likely that these fibroblast lineages play unique roles during epithelial cancer formation and progression. Therefore, I would like to focus my research on unravelling the mechanism by which cancer cells reprogram normal fibroblasts into cancer-associated fibroblasts, and how CAFs alter the microenvironment to promote tumour growth and metastasis in particular in squamous cell carcinoma of the skin and melanoma, which has a high predilection for metastatic progression with 44-64% of patients developing brain metastasis. By employing in vivo lineage tracing techniques in combination with single cell RNA sequencing I aim to disclose the origin of CAFs and dissect stromal heterogeneity at both molecular and functional levels, and address how different CAF subsets support malignant progression. In conclusion, this study will not only improve our understanding about the role of the tumour stroma and microenvironment during tumour formation and metastasis but will also provide novel targets for anti-tumour therapy, new markers which could be used for drug delivery via immunotherapy, as well as biomarkers for evaluating the metastatic potential of a given tumour, and might also boost the development of personalized cancer treatments.
publications:
Cellular heterogeneity and microenvironmental control of skin cancer. Lichtenberger BM and Kasper M. JIM 2020 Sept, PMID: 32976658
Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting Korosec A, Frech S, Lichtenberger BM. JoVE 2019 May, PMID: 31132050
Lineage identity and location within the dermis determine the function of papillary and reticular fibroblasts in human skin. Korosec A, Frech S, Gesslbauer B, Vierhapper M, Radtke C, Petzelbauer P, Lichtenberger BM. J Invest Dermatol. 2018 Sep, PMID: 30179601
CAF variants control the tumor-immune microenvironment and predict skin cancer malignancy Forsthuber A, Korosec A, Jacob T, Aschenbrenner B, Annusver K, Frech S, Purkhauser K, Krajic N, Lipp K, Werner F, Nguyen V, Griss J, Bauer W, Soler Cardona A, Weber B, Weninger W, Gesslbauer B, Staud C, Nedomansky J, Radtke C, Wagner SN, Petzelbauer P, Kasper M, Lichtenberger BM. doi: https://doi.org/10.1101/2023.05.03.539213
project 2) “Functional Deconstruction of Fibroblast Heterogeneity in Malignant Skin Cancer” funded by ÖNB
approved amount (1.8.2018 – 30.7.2021):
€ 148.000
summary:
In a complementary study funded by the ÖNB we decipher distinct CAF subsets and their crosstalk with cancer cells in human BCC, cSCC and melanoma using single cell RNA sequencing.
publications:
Cellular heterogeneity and microenvironmental control of skin cancer. Lichtenberger BM and Kasper M. JIM 2020 Sept, PMID: 32976658
CAF variants control the tumor-immune microenvironment and predict skin cancer malignancy Forsthuber A, Korosec A, Jacob T, Aschenbrenner B, Annusver K, Frech S, Purkhauser K, Krajic N, Lipp K, Werner F, Nguyen V, Griss J, Bauer W, Soler Cardona A, Weber B, Weninger W, Gesslbauer B, Staud C, Nedomansky J, Radtke C, Wagner SN, Petzelbauer P, Kasper M, Lichtenberger BM. doi: https://doi.org/10.1101/2023.05.03.539213
project 3) “Molecular Atlas of human skin cancer” funded by the City of Vienna Fund for Innovative Interdisciplinary Cancer Research
approved amount (02.2020 – 01/2021):
€ 39.551
summary:
Although fibroblasts have originally been considered an underwhelming cell type, they have important functions in tissue development, homeostasis, regeneration and also in pathological conditions and aging, and we are just appreciating the high cell plasticity and heterogeneity within the skin mesenchyme. Recent studies revealed that mouse skin dermis arises from two distinct fibroblast lineages with different functions in skin homeostasis, regeneration and fibrosis. Accordingly, data from the lab and others suggest that also human skin contains several functionally distinct fibroblast subsets. Supposedly, these fibroblast lineages also play unique roles in epithelial cancers, incidences and death rates of which have been increasing at an alarming rate, especially within the younger population, thus elevating malignant skin cancer to a profound public health concern. Studies in various organs and cancer types suggest that the so-called cancer-associated fibroblasts (CAFs) differ from fibroblasts in healthy tissue and play important roles in malignant cancer progression. However, the origin of CAFs and their phenotypic and functional heterogeneity remain elusive, particularly in malignant cutaneous cancer.
We use single-cell RNA sequencing (scRNA-seq) and mass cytometry-based tissue imaging to molecularly profile the full repertoire of cells in human healthy skin, basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC) and melanoma. Utilising a state-of-the-art computational analysis pipeline, we aim at identifying specific fibroblast subsets and dissecting their direct or paracrine interplay with (i) neoplastic epidermal cells, (ii) immune cells and (iii) other stroma cell. We are specifically interested in the immuno-modulatory role of fibroblasts that might result in immune-suppression and check-point inhibitor treatment resistance. Based on the scRNA-seq data, we will develop a panel of biomarkers for mass cytometry imaging to confirm cell interactions and the signalling pathways involved in situ, and most importantly for routine diagnostic and prognostic analysis of paraffin-embedded cutaneous cancer tissue in order to establish personalized therapies.
By establishing a cellular and molecular map of human BCC, cSCC and melanoma, this study will lead to major advances in our understanding of fundamental mechanisms resulting in skin pathologies, and provide new insights into skin fibroblast heterogeneity and plasticity and how specific CAF subsets support malignant progression. Furthermore, our findings will reveal novel targets for anti-tumour therapy as well as panels of biomarkers for evaluating the metastatic potential of a given tumour and prognostic assessment, and may thus boost the development of personalized cancer treatments.
publications:
Cellular heterogeneity and microenvironmental control of skin cancer. Lichtenberger BM and Kasper M. JIM 2020 Sept, PMID: 32976658
CAF variants control the tumor-immune microenvironment and predict skin cancer malignancy Forsthuber A, Korosec A, Jacob T, Aschenbrenner B, Annusver K, Frech S, Purkhauser K, Krajic N, Lipp K, Werner F, Nguyen V, Griss J, Bauer W, Soler Cardona A, Weber B, Weninger W, Gesslbauer B, Staud C, Nedomansky J, Radtke C, Wagner SN, Petzelbauer P, Kasper M, Lichtenberger BM. doi: https://doi.org/10.1101/2023.05.03.539213
project 4) “Imaging Mass Cytometry for diagnostic and prognostic analysis of skin cancer” funded by the City of Vienna Fund for Innovative Interdisciplinary Cancer Research (GMWF-21059)
approved amount (06.2021 – 09/2022):
€ 40.000
summary:
The incidence and death rates of epithelial cancers have been increasing at an alarming rate, especially within the younger population, thus elevating malignant skin cancer to a profound public health concern. In recent years, strong evidence accumulated that the immediate environment of mutant cells profoundly impact their prospect of malignant progression. Studies in various organs and cancer types suggest that the so-called cancer-associated fibroblasts (CAFs) play important roles in cancer development and progression, and that several subsets exist which can be both tumor-promoting and -suppressing. To deconstruct the contribution of CAF subsets in skin cancer, we used single-cell RNA sequencing (scRNA-Seq) to molecularly profile the full repertoire of cells in human healthy skin, basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC) and melanoma. Utilising a state-of-the-art computational analysis pipeline, we identified seven specific fibroblast subsets and dissected their direct or paracrine interplay with (i) neoplastic cells, (ii) immune cells and (iii) other stroma cells. Intriguingly, the expression profile of CAF subsets that are predominant in malignant cancers predict an immuno-modulatory function that might result in immune-suppression and check-point inhibitor treatment resistance. Based on the scRNA-Seq data, we will develop a panel of biomarkers for RNAscope in situ hybridisation and imaging mass cytometry (IMC). This will allow a rapid in situ analysis of human paraffin-embedded tumor samples aiming at the establishment of a cellular and molecular map of human skin cancer and its associated fibroblast subsets. Matching the results with clinical data (tumor progression, survival and response to therapy), this will provide a novel tool for routine diagnostic analysis of paraffin-embedded cutaneous tumor tissues to predict patient prognosis and the basis for personalised therapeutic decisions.
project 5) “Controlling the epigenetic switch of skin fibroblast aging” funded by the Austrian Science Fund (FWF, P25207)
approved amount (02.2022 – 01/2025):
€ 382.958
summary:
The aging-skin fragility, infections, impaired wound healing, and skin cancer. Aging-dependent skin alterations are associated with significant changes of the connective tissue and dermal fibroblasts, an important cell type that keeps our skin smooth and elastic. Fibroblasts display an unprecedented heterogeneity and plasticity, but how fibroblast fate decisions are controlled is unknown. We aim at investigating if their differentiation programs in skin homeostasis and aging are controlled epigenetically. Identification of the epigenetic switches regulating the balance between fibroblast proliferation, differentiation and senescence could help to counteract skin aging and associated defects in wound healing. We will perform Single-cell-Assay-for-Transposase-Accessible-Chromatin (scATAC) and single-cell-RNA sequencing (scRNA-Seq) of fibroblasts isolated from human skin of young and old donors followed by a range of in-vitro and in-vivo assays to determine the epigenome and transcriptome of young and aged, pathogenic fibroblasts. Our studies will unravel if fibroblast differentiation programs are controlled by reversible epigenetic marks, and reveal if targeting these epigenetic switches by pharmacological drugs could be used to resolve aging-mediated defects of skin function, for example by reprogramming senescent or fibrotic fibroblasts, thereby rejuvenating the skin dermis and improving wound repair, especially in the elderly.
project 6) “Skin cancer organoids for personalized drug screening” funded by the City of Vienna Fund for Innovative Interdisciplinary Cancer Research (GMWF-22059)
approved amount (06.2022 – 06/2024):
€ 78,225
summary:
Patient-derived cancer organoids (PDOs) are excellent in vitro models that precisely recapitulate the pathophysiological nature of tumorigenesis. Maintaining the cellular heterogeneity of the original tumor provides a unique opportunity to dissect complex cancer cell-stroma interactions, and to advance cancer research and precision medicine.
PDOs have been established from various types of cancers, however not from cutaneous squamous cell carcinoma (cSCC). Many of these PDOs were generated by submerged culture, which typically solely involves epithelial cells. By employing air–liquid interface (ALI) culture methodology, we aim at establishing cancer organoids from human cSCC biopsies comprising both cancer and stroma cells (i) to develop an in vitro drug screening pipeline for novel targets as well as for the development of personalized cancer treatments, and (ii) to dissect the interactions of distinct cancer-associated fibroblast (CAF) subsets with tumor and immune cells, since single cell RNA sequencing (scRNA-Seq) predicts distinct functions for 4 CAF subsets in cSCC according to their expression profiles. Intriguingly, one CAF subset might affect immune surveillance and the response to immunotherapy, which we aim at dissecting in PDOs. The quality and cellular composition of the organoids will be assessed by FACS-analysis, immunofluorescent stainings (with various cell-type specific markers) and confocal microscopy. Single cell RNA sequencing (scRNA-Seq; SmartSeq3) of cancer organoids and their corresponding native tumors will be performed with our established protocols for skin cancer biopsies to assess if the cellular and molecular composition of the cancer organoids is comparable to native tumors. In addition, assays for high throughput drug screening in PDOs will be developed.
project 7) “Replacing in vivo skin cancer studies by tumor organoids” funded by the Austrian Science Fund (FWF, P36368)
approved amount (03/2023 – 02/2026):
€ 385.838
abstract:
Preclinical cancer research has relied on animal models and on the analysis of immortalized two-dimensional (2D) cancer cell lines in vitro for decades. Although animal models are time-consuming and costly, and cancer cell lines may not recapitulate the pathophysiological features of the original tumor, these two approaches have tremendously contributed to our understanding of cancer development and progression, and have led to a multitude of different cancer therapies. In recent years novel 3D cancer organoid cultures have been developed, which combine the experimental feasibility and tractability of cancer cell lines with the complex cellular heterogeneity of in vivo model systems. These patient-derived cancer organoids (PDOs) are excellent in vitro models that precisely recapitulate the pathophysiological nature of tumorigenesis. Cancer organoids have been established from various types of cancers, however not from human cutaneous squamous cell carcinoma (cSCC). Many of these PDOs were generated by submerged culture, which typically solely involves epithelial cells. We will employ airliquid interface (ALI) culture methodology to generate PDOs that include epithelial cells alongside integrated stroma and immune cells, thereby allowing to assess the contribution of the tumor stroma to cancer growth and treatment response. Specifically, we aim at establishing cancer organoids from human cSCC biopsies comprising both cancer and stroma cells (i) to replace in vivo skin cancer studies in animals, (ii) to develop an in vitro drug screening pipeline for novel targets as well as for the development of personalized cancer treatments, and (iii) to dissect the interactions of distinct cancer-associated fibroblast (CAF) subsets with tumor and immune cells, since single-cell RNA sequencing (scRNA-Seq) predicts distinct functions for three CAF subsets in cSCC according to their expression profiles. Intriguingly, one CAF subset might affect immune surveillance and the response to immunotherapy, which we aim at dissecting in PDOs. Maintaining the cellular heterogeneity of the original tumor provides a unique opportunity to dissect complex cancer cell-stroma interactions, and to advance cancer research and precision medicine.velopment of personalized cancer treatments.