While triple-negative breast cancer (TNBC) is known to be heterogeneous at the genomic and transcriptomic levels, spatial information on tumor organization and cell composition is still lacking. Here, we investigate TNBC tumor architecture including its microenvironment using spatial transcriptomics on a series of 92 patients. We perform an in-depth characterization of tumor and stroma organization and composition using an integrative approach combining histomorphological and spatial transcriptomics. Furthermore, a detailed molecular characterization of tertiary lymphoid structures leads to identify a gene signature strongly associated to disease outcome and response to immunotherapy in several tumor types beyond TNBC. A stepwise clustering analysis identifies nine TNBC spatial archetypes, further validated in external datasets. Several spatial archetypes are associated with disease outcome and characterized by potentially actionable features. In this work, we provide a comprehensive insight into the complexity of TNBC ecosystem with potential clinical relevance, opening avenues for treatment tailoring including immunotherapy.

Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins. 

Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer(1). Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics(2) to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.

Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3′ end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method's capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method's potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery.

In the present moment of time, we find ourselves in a period where the advancement of genomic tools is progressing at a fast pace. Of particular interest for this thesis is the study of gene activity. What patterns of genes are expressed? Where are they expressed? How can we use this knowledge to improve our quality of life? The research presented in this thesis focuses on developing and applying new tools for interrogating cells and tissues. In Paper I, we describe a protocol for transcript profiling of single cells, capable of measuring the relative expression levels for genes of interest. We successfully applied our method to cancer cells from metastatic breast cancer patients. Profiling 2 to 4 single cells per patient and measuring gene-specific expression from targets previously associated with metastatic breast cancer supports the use of our protocol as a diagnostic tool. In Paper II, we present an assay for spatial RNA quality evaluation, used to estimate the success for tissue specimens before proceeding with more expensive spatial sequencing methods. We showed that the method is capable of measuring high RNA quality in tissue areas of both high and low cell density and that the spatial RNA integrity patterns are reflected in spatial transcriptomics data. In Paper III, we present a protocol for performing spatial mRNA genome-wide expression profiling of FFPE tissue specimens. Thus, we bridge a gap between traditional tissue preservation methods and novel gene technologytools. We found a high Pearson correlation of 0.95 between formalin-fixation paraffin embedding (FFPE) and Fresh Frozen (FF) mouse brain datasets. Although the FPPE samples yielded fewer transcripts and genes compared to FF, there was a high agreement in gene expression between paired anatomical areas for FFPE and FF samples. In Paper IV, we present an approach to investigate in situ transcript derivedinferred copy number variation (iCNV) profiles based on spatial transcriptomics data. In a normal lymph node that displays both distinct gene expression patterns and histological landmarks, we observed a neutral iCNV profile. In contrast, we found huge variabilities investigating several malign tissue types ranging from homogenous (pediatric medulloblastoma) to highly variable genomes (ductal breast cancer and glioblastoma). Strikingly, we also observed similar iCNV profiles in both tumor and benign tissue areas from prostate and skin cancer. In Paper V, we explore the transcriptional and genomic landscape in pediatric tumors from 14 patients. Microglia cells have been implicated to play an important role in the tumor microenvironment, and we found spatial co-localization of microglia and epithelial-to-mesenchymal transition (EMT) signatures in our patient cohort. Furthermore, we found homogenous and recurrent iCNV profiles in the high-grade tumors of relapse patients and identified expression of gene SPP1 in the tumor stroma as a potential prognostic mRNA marker in pediatric brain tumor relapse patients.

Vi befinner oss i en tid där framsteg för genomiska verktyg fortskrider i snabb takt. Av särskilt intresse för denna avhandling är studier av genaktivitet. Vilka genmönster uttrycks? Var uttrycks de? Hur kan vi använda denna kunskap för att förbättra vår livskvalitet? Forskningen som presenteras i denna avhandling fokuserar på att utveckla och tillämpa nya verktyg för att inhämta information från celler och vävnader. I Artikel I beskriver vi ett protokoll för profilering av transkript i enstaka celler som kan mäta de relativa uttrycksnivåerna för gener av intresse. Vi har framgångsrikt tillämpat vår metod på cancerceller från metastatiska bröstcancerpatienter. Profilering utfördes på 2 till 4 celler per patient och uppmätte ett genspecifikt uttryck från markörer som tidigare varit associerade med metastatisk bröstcancer. Dessa resultat stöder användningen av vårt protokoll som ett diagnostiskt verktyg. I Artikel II presenterar vi en analys för utvärdering av spatial RNA-kvalitet, som används för att estimera integriteten av vävnadsprover innan dyrare spatiala sekvenseringsmetoder appliceras. Vi visade att metoden kan uppmäta hög RNA-kvalitet i vävnadsområden med både hög och låg celltäthet, samt att de spatiala RNA-integritetsmönstren återspeglas i spatial transkriptomikdata. I Artikel III presenterar vi ett protokoll för att utföra spatial mRNA genom profilering av FFPE-vävnadsprover. Således sammanlänkar vi traditionella vävnadsbevaringsmetoder med nya gentekniska verktyg. Vi observerade en hög Pearson-korrelation på 0.95 mellan formalinfixerade paraffininbäddade (FFPE) och färskfrysta (FF) datasett från mushjärna. Även om ett mindre antal transkript och gener kunde utvinnas från FFPE jämfört med FF prover, fanns det en hög överensstämmelse i genuttryck mellan parade anatomiska områden för FFPE- och FF-prover. I Artikel IV presenterar vi ett tillvägagångssätt för att undersöka variationer i inferrerade spatiala genkopior (iCNV) inom vävnadssnitt, baserat på spatial transkriptomikdata. I normal lymfkörtel som visar både distinkta genuttrycksmönster och histologiska landmärken observerade vi en neutral iCNV - profil. Däremot fann vi stora variationer från homogena (pediatriska medulloblastom) till mycket variabla genom (duktal bröstcancer och glioblastom) när vi undersökte flera maligna vävnadstyper. Påfallande nog observerade vi också liknande iCNV -profiler i både tumör- och godartade vävnadsområden från prostata och hudcancer. I Artikel V utforskar vi det transkriptionella och genomiska landskapet i pediatriska tumörer från 14 patienter. Microglia-celler har implicerats att spela en viktig roll i tumörmikromiljön, och vi fann spatial samlokalisering av mikroglia och epitel-till-mesenkymal övergångs (EMT) signaturer i vår patientgrupp. Vidare hittade vi homogena och återkommande iCNV-profiler i höggradiga tumörer hos återfallspatienter och identifierade genuttryck av SPP1 inom tumörstroman som en potentiell prognostisk mRNA-markör hos pediatriska patienter med återfall av hjärntumör.

The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows. Kvastad et al. develop the spatial RNA Integrity Number (sRIN) assay that evaluates the RNA integrity at cellular resolution. This method improves the resolution of a similar method called the RNA Integrity Number (RIN), demonstrating spatial variation in the quality of RNA samples.

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.